UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The adherence of Acidiphilium cryptum to chalcopyrite Heffelfinger, Blair 1990

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1990_A6_7 H43.pdf [ 7.49MB ]
Metadata
JSON: 831-1.0098025.json
JSON-LD: 831-1.0098025-ld.json
RDF/XML (Pretty): 831-1.0098025-rdf.xml
RDF/JSON: 831-1.0098025-rdf.json
Turtle: 831-1.0098025-turtle.txt
N-Triples: 831-1.0098025-rdf-ntriples.txt
Original Record: 831-1.0098025-source.json
Full Text
831-1.0098025-fulltext.txt
Citation
831-1.0098025.ris

Full Text

The adherence of Acidiphilium cryptum to chalcopyrite By B l a i r H e f f e l f i n g e r B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1 9 8 5 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF MICROBIOLOGY We accept t h i s t h e s i s as conforming t o the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA January 1 9 9 0 © B l a i r H e f f e l f i n g e r In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree th a t permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada Date DE-6 (2/88) \ ABSTRACT A c i d i p h i l i u m cryptum i s a h e t e r o t r o p h i c a c i d o p h i l e commonly found i n a c i d i c environments and i n d u s t r i a l b i o l e a c h i n g operations. Attachment t o mineral surfaces may serve to maintain t h i s organism i n aqueous environments where i t i s subject t o removal by hydrodynamic f o r c e s . Using i n d i r e c t and d i r e c t methods we have looked at the b i n d i n g of A.cryptum to c h a l c o p y r i t e (CuFeS2) and other mineral ores t o determine whether s p e c i f i c adhesins mediate b i n d i n g . A modified ELISA b i n d i n g assay (the Ore ELISA) was developed to measure d i r e c t adherence. F i n e l y ground c h a l c o p y r i t e was bound i r r e v e r s i b l y to the w a l l s of an ELISA p l a t e , the organisms were added and a f t e r i n c u b a t i o n and washing, the number of attached b a c t e r i a were assessed by r e a c t i n g w i t h anti-A.crypturn antibody followed by goat a n t i - r a b b i t IgG conjugated t o a l k a l i n e phosphatase. This assay was found t o be s e n s i t i v e , r a p i d and r e p r o d u c i b l e . The Ore ELISA allowed d i r e c t b i n d i n g measurement i n the presence of v a r i o u s i n h i b i t o r s and provided a r a p i d screening method f o r adherence-defective mutants. Adherence was shown to be satu r a b l e and increased s l i g h t l y as pH decreased. A moderate increase i n b i n d i n g a f f i n i t y was recorded i n the presence of monovalent and d i v a l e n t c a t i o n s and EDTA. Various b a c t e r i c i d a l agents and pentose and hexose sugars had no e f f e c t on c h a l c o p y r i t e attachment. Reducing agents had l i t t l e e f f e c t on c e l l adherence. A strong increase i n i i adherence was observed i n the presence of surface a c t i v e agents. Bovine serum albumin and g e l a t i n were both found to markedly reduce mineral surface b i n d i n g . Competition f o r attachment s i t e s between A.cryptum and the aut o t r o p h i c a c i d o p h i l e , Thiobacillus ferrooxidans, showed that each organism binds to unique s i t e s on the c h a l c o p y r i t e surface. A.cryptum mutant s t r a i n s d i s p l a y i n g reduced adherence to c h a l c o p y r i t e were shown to l a c k a 31.6 kDa outer membrane p r o t e i n . i i i T A B L E O F C O N T E N T S I n t r o d u c t i o n 1 A c i d o p h i l e s 1 Adherence 19 M a t e r i a l s and Methods 37 B a c t e r i a 37 Media and stock maintenance 37 B u f f e r s 38 Bio-Rad adherence assay 38 Ore ELISA adherence assay 39 I s o l a t i o n of adherence v a r i a n t s 42 C e l l envelope p r o t e i n p r e p a r a t i o n 43 C e l l envelope s o l u b i l i z a t i o n 43 I n h i b i t i o n assay 44 C e l l m o d i f i c a t i o n 44 Competition assay 45 Hydrophobicity assay 45 Surface p r o t e i n p r e p a r a t i o n (SPP) 46 SDS-Polyacrylamide g e l e l e c t r o p h o r e s i s 46 Pre p a r a t i o n of IgG 47 Adsorption of IgG 47 Western b l o t 48 i v Immunogold bead l a b e l l i n g 4 9 E l e c t r o n microscopy 49 SPP i n h i b i t i o n assay 49 Antibody i n h i b i t i o n assay 50 Results 51 Bio-Rad assay f o r measuring A.cryptum adherence to c h a l c o p y r i t e 51 Development of the Ore ELISA assay 57 Adherence i n h i b i t i o n s t u d i e s 65 Competition stud i e s 78 I s o l a t i o n of adherence-defective v a r i a n t s 81 A n a l y s i s of adherence-defective mutants 85 L o c a l i z a t i o n of the 31.6 kDa p r o t e i n 87 D i s c u s s i o n 100 B i b l i o g r a p h y 109 v LIST OP TABLES v i LIST OF FIGURES Figure Page 1 Bio-Rad assay i l l u s t r a t i n g the adsorption of A.cryptum to c h a l c o p y r i t e 52 2 Bio-Rad assay demonstrating the r e l a t i o n s h i p between the number of c e l l s bound and the amount of Newmont added 53 3 Bio-Rad assay i l l u s t r a t i n g c e l l adsorption as a f u n c t i o n of adherence b u f f e r pH 54 4 Bio-Rad assay i l l u s t r a t i n g the r e l a t i o n s h i p between i n c u b a t i o n time and the number of A.cryptum c e l l s bound 55 5 Cr o s s - s e c t i o n of an Ore ELISA p l a t e 58 6 E f f e c t of assay pH on adsorption at 405 nm 59 7 Ore ELISA measuring the adsorption isotherm f o r A.cryptum b i n d i n g t o c h a l c o p y r i t e 66 8 Ore ELISA demonstrating the e f f e c t of adherence b u f f e r pH on A.cryptum b i n d i n g t o c h a l c o p y r i t e 67 9 R e l a t i o n s h i p between length of in c u b a t i o n and adherence as measured by the Ore ELISA assay 68 10 Adherence to d i f f e r e n t minerals 69 11 Ore ELISA measuring the bi n d i n g of T.ferrooxidans to c h a l c o p y r i t e 79 12 Competition between A.cryptum and T.ferrooxidans f o r b i n d i n g s i t e s on c h a l c o p y r i t e 80 13 Nitrosoguanidine s u r v i v a l curve 82 v i i 14 Western b l o t of SDS-PAGE c o n t a i n i n g p a r e n t a l and mutant whole c e l l l y s a t e s developed w i t h anti-A.cryptum antibody 86 15 SDS-PAGE of surface p r o t e i n preparations 88 16 Western b l o t of SDS-PAGE c o n t a i n i n g surface p r o t e i n preparations developed w i t h a n t i -A.cryptum antibody 89 17 Western b l o t of SDS-PAGE c o n t a i n i n g CEP preparations developed w i t h anti-31.6 kDa antibody 92 18 Immunogold bead l a b e l l i n g of p a r e n t a l A.cryptum w i t h anti-31.6 kDa antibody 94 19(a) Immunogold bead l a b e l l i n g of mutant-15 wi t h anti-31.6 kDa antibody 95 19(b) Immunogold bead l a b e l l i n g of p a r e n t a l A.cryptum w i t h non-immune serum 96 20 Immunogold bead l a b e l l i n g of p a r e n t a l A.cryptum w i t h anti-31.6 kDa antibody ( d i s r u p t i v e ) 97 21 Immunogold bead l a b e l l i n g of a p a r e n t a l s t r a i n A.cryptum c e l l p r i n t w i t h anti-31.6 kDa antibody 98 v i i i Dedicated to my w i f e , L e s l e y . i x ACKNOWLEDGEMENTS I t h a n k f u l l y acknowledge the support, encouragement and guidance of Dr. B. C. McBride. I a l s o g r a t e f u l l y acknowledge the f i n a n c i a l support of the U n i v e r s i t y of B r i t i s h Columbia and the Medical Research C o u n c i l of Canada. I extend my thanks to Drs. D. K i l b u r n and J . Smit f o r t h e i r guidance wh i l e s e r v i n g on my committee. I wish to thank Angela Joe, Drs. Daniel Grenier, Umadatt Singh and Nadarajah Ganeshkumar f o r s h a r ing t h e i r t e c h n i c a l e x p e r t i s e . I g r a t e f u l l y acknowledge Pauline Hannam f o r her as s i s t a n c e during some of the experiments i n c l u d e d i n t h i s document. I o f f e r my s i n c e r e thanks t o Laura W i l l i a m s , Dr. Helen Scott and Heather M e r i l e e s f o r t h e i r support and sense of humour. I would a l s o l i k e t o thank my f r i e n d s and fa m i l y f o r t h e i r encouragement, Andre Wong f o r h i s work w i t h the e l e c t r o n microscope and Bruce McCaughey and Harold Traeger f o r t h e i r photography. I extend s p e c i a l thanks to Lesley H e f f e l f i n g e r f o r a s s i s t i n g i n the pr e p a r a t i o n of t h i s manuscript. x INTRODUCTION Acidophiles A c i d o p h i l e s are unique among microorganisms i n that they l i v e at pHs below 5.0. A c i d o p h i l e s can be d i v i d e d i n t o two groups; f a c u l t a t i v e a c i d o p h i l e s and extreme a c i d o p h i l e s . F a c u l t a t i v e a c i d o p h i l e s , which are capable of growth at both n e u t r a l and a c i d i c pH, in c l u d e such b a c t e r i a l species as Streptococcus, Lactobacillus, and some species of Escherichia and Staphylococcus. Extreme a c i d o p h i l e s r e q u i r e an a c i d i c environment f o r growth and w i l l p e r i s h at n e u t r a l pH. Heterotrophic extreme a c i d o p h i l e s use a v a r i e t y of carbohydrates and organic acids as both an energy and carbon sources. Examples of h e t e r o t r o p h i c a c i d o p h i l e s are Acidiphilium spp. and Thiobacillus acidophilus. A u t o l i t h o t r o p h i c extreme a c i d o p h i l e s o b t a i n energy through the o x i d a t i o n of reduced i r o n and sulphur compounds and carbon through the f i x a t i o n of carbon d i o x i d e . Examples of aut o t r o p h i c a c i d o p h i l e s i n c l u d e Thiobacillus ferrooxidans and Thiobacillus thiooxidans, which o x i d i z e reduced s u l p h u r / i r o n and sulphur r e s p e c t i v e l y (Brock, 1979). The most common source of a c i d o p h i l i c microorganisms i s a c i d mine drainage (AMD) waters. Since t h e i r i s o l a t i o n and p o s t u l a t e d involvement i n AMD (Colmer, 1950), T h i o b a c i l l u s ferrooxidans has been the subject of considerable environmental research. AMD i s c h a r a c t e r i z e d by, a pH below 4.5, high f e r r i c i r o n and sulphate 1 l e v e l s , low concentrations of organic carbon, high l e v e l s of aluminum, calcium ions, s o l u b l e heavy metals (manganese and l e a d ) , and reduced l e v e l s of phosphate and n i t r o g e n (Johnson et a l . , 1979). There are two primary sources of mine waste: (1) T a i l i n g s , which are the by-products of mineral ore m i l l i n g and separation; (2) Overburden, which i s the s o i l and r e s i d u a l ore m a t e r i a l l e f t at the mine s i t e . Both forms of mine waste have s i g n i f i c a n t concentrations of reduced sulphur compounds which are o x i d i z e d through m i c r o b i a l a c t i o n to form AMD ( M i l l s , 1985). With the dwindling reserves of high grade ore and an i n c r e a s i n g demand f o r f o s s i l f u e l s , most fut u r e m e t a l - r e f i n i n g and f o s s i l f u e l production w i l l i n v o l v e surface s t r i p mining, r e s u l t i n g i n increased water p o l l u t i o n through mine drainage. The e f f e c t s of AMD on the environment are w e l l documented (Carpenter, 1925; Campbell and Lind , 1969; Yan, 1979; Johnson et a l . , 1978; N o r r i s et a l . , 1981; Olem, 1981). Most repo r t s have st u d i e d the physicochemical e f f e c t on the environment; the n a t u r a l f l o r a , plankton, v e g e t a t i o n , i n v e r t e b r a t e s and f i s h . The e f f e c t s of AMD on the m i c r o b i a l community has l a r g e l y been l i m i t e d to i r o n and sulphur o x i d i z i n g organisms such as T.ferrooxidans and T h i o b a c i l l u s thiooxidans ( B r i e r l e y , 1978). There i s a growing i n t e r e s t i n the e f f e c t s of AMD on h e t e r o t r o p h i c organisms and t h e i r c o n t r i b u t i o n , i f any, to the r e h a b i l i t a t i o n of a c i d mine p o l l u t e d water. In general most 2 h e t e r o t r o p h i c microbes are s e n s i t i v e to pH v a l u e below 5 or above 9 (Alexander , 1977) . In a d d i t i o n , meta ls such as Cu , C d , Pb and Zn, which are found i n h i g h l e v e l s i n AMD, are t o x i c t o many microorgani sms ( S t e r r i t t & L e s t e r , 1980; Zevenhuizen et a l . , 197 9) . Though c o n s i d e r a b l e r e s e a r c h has q u e s t i o n e d the e f f e c t s o f c e r t a i n a spec t s o f AMD on m i c r o o r g a n i s m s , c o m p a r a t i v e l y l i t t l e work has addres sed the m i c r o b i a l response and s u c c e s s i o n o f events f o l l o w i n g exposure t o AMD. Wassel e t a l . ( 1 9 8 3 ) s t u d i e d the water and sediment b a c t e r i a l communit ies f o r s t r e s s - r e l a t e d responses to a p o i n t source o f a c i d mine d r a i n a g e . In g e n e r a l , they found t h a t the d i v e r s i t y o f the m i c r o b i a l communit ies was s i g n i f i c a n t l y lower at the s i t e r e c e i v i n g AMD as compared to uncontaminated c o n t r o l s ; microorgan i sms at the contaminated s i t e s were more s i m i l a r t o each o t h e r than they were to the c o n t r o l s . M i l l s s u p p o r t e d these f i n d i n g s i n c o n c l u d i n g "that m i c r o b i a l communit ies d e v e l o p i n g i n extreme environments t e n d to be g e n e r a l i s t s , where as those i n mesic environments t e n d t o be s p e c i a l i s t s " ( M i l l s & M a l l o r y , 1987) . U n l i k e the a u t o t r o p h i c Thiobacillus s p p . , h e t e r o t r o p h i c b a c t e r i a were l o n g c o n s i d e r e d t r a n s i e n t s i n a c i d mine waters ( T u t t l e et a l . , 1968) . However, s p e c i a l l y adapted h e t e r o t r o p h i c organisms have been i s o l a t e d and are capab le o f growth i n AMD p o l l u t e d waters (Wichlacz e t a l . , 1981; Manning, 1975) . AMD i s not the o n l y source o f a c i d o p h i l i c h e t e r o t r o p h s , they have a l s o been i s o l a t e d from a c i d i c sewage and s o i l , and from what was presumed 3 t o be pure c u l t u r e s of T.ferrooxidans (Kishimoto, 1987; Har r i s o n , 1980; Zavarzin, 1972; Johnson & Kelso, 1983). Through DNA homology s t u d i e s , 25% of 23 s t a i n s of T.ferrooxidans were found to contain h e t e r o t r o p h i c contaminants (Harrison, 1982). One common contaminant was Thiobacillus acidophilus which i s described by Arkesteyn et a l . (1980) as a mixotrophic T h i o b a c i l l u s species. A second common contaminant contained a DNA G:C content (68-70%) beyond the range of the Thiobacillus species. H a r r i s o n et a l . (1981), proposed a new generic name, Acidiphilium gen. nov. (type species: Acidiphilium cryptum) and described i t as a m o t i l e , gram-negative, aerobic, mesophyllic, rod-shaped, h e t e r o t r o p h i c a c i d o p h i l e . This new a c i d o p h i l e was non-encapsulated, m o t i l e by a p o l a r or two l a t e r a l f l a g e l l u m and unable to o x i d i z e reduced sulphur or i r o n compounds. A.cryptum can u t i l i z e v a r i o u s carbohydrates as sol e carbon and energy sources. Operational pentose phosphate and Entner-Doudoroff pathways were i d e n t i f i e d by Shuttleworth et a l . (1985). Since i t s discovery i n 1981, four a d d i t i o n species of A c i d i p h i l i u m have been i s o l a t e d ; Acidiphilium angustum, Acidiphilium f a c i l i s , Acidiphilium rubrum (Wichlacz, 1986) and Acidiphilium organovorum (Lobos, 1986) . I t has been suggested that the main cause of v a r i a b l e o x i d a t i o n rates between d i f f e r e n t T. ferrooxidans c u l t u r e s ( K e l l y & Jones, 1978; Chang and Myerson, 1982; Roy et a l . , 1981; Gormely et a l . , 1975) i s a t t r i b u t e d t o h e t e r o t r o p h i c contaminants. H a r r i s o n et 4 a l . , suggested t h a t since A c i d i p h i l i u m and T h i o b a c i l l u s are so t e n a c i o u s l y a s s o c i a t e d i n c u l t u r e d i s o l a t e s , they may cross-feed i n the n a t u r a l environment (Harrison, 1980). The idea of m i c r o b i a l symbiosis between l i t h o t r o p h s and heterotrophs i s not new. I t i s commonly found, i n b i o l e a c h i n g operations, that mixed c u l t u r e s of b a c t e r i a have a higher mineral y i e l d than pure c u l t u r e of a u t o t r o p h i c organisms (Andrews et a l . , 1982; Detz, 197 9; Hoffman et a l . , 1981; Dugan, 1978; Andrews et a l . , 1987; N o r r i s & K e l l y , 1978). Many simple organic compounds have been found to i n h i b i t growth and i r o n and sulphur o x i d a t i o n by T. ferrooxidans ( T u t t l e & Dugan, 1976; Rao & Berger, 1970). Arkesteyn (1980) demonstrated that i n h i b i t i o n of i r o n - o x i d i z i n g T. ferrooxidans by c e r t a i n organic compounds (eg. 1.0 mM Glucose, ethanol, l a c t a t e , succinate, s e r i n e and aspartate) was r e l i e v e d when the mixotrophic T.acidophilus was added to the a u t o t r o p h i c T.ferrooxidans. A m u t u a l i s t i c r e l a t i o n s h i p i s b e l i e v e d t o occur between the i r o n / s u l p h u r o x i d i z i n g T.ferrooxidans and the s t r i c t l y sulphur o x i d i z i n g T.thiooxidans. As i r o n o x i d a t i o n proceeds, sulphur l a y e r s may coat the ore substrate and i n h i b i t f u r t h e r o x i d a t i o n . T.thiooxidans i s b e l i e v e d to o x i d i z e the accumulating sulphur and thus expose more ferrous substrate f o r T.ferrooxidans, however, general agreement on t h i s theory i s l a c k i n g . K e l l y et a l . i n 1978, described a symbiotic r e l a t i o n s h i p between the i r o n o x i d i z i n g a c i d o p h i l e , Leptospirillium ferrooxidans and the sulphur o x i d i z i n g Thiobacillus organoparus ( K e l l y et a l . , 1978). 5 The mechanisms by which organic compounds i n h i b i t T. ferrooxidans i s p o o r l y understood. Using e l e c t r o n micrographs, T u t t l e et a l . (1977), i l l u s t r a t e d c e l l envelope d i s r u p t i o n when T. ferrooxidans was exposed t o organic a c i d s . In 1970, Rao and Berger demonstrated passive p e r m e a b i l i t y to p y r u v i c a c i d at low pH, r e s u l t i n g i n a decreased i n t r a c e l l u l a r pH. Ingledew proposed that the t o x i c i t y of weak acids can be a t t r i b u t e d to t h e i r accumulation i n the c e l l matrix i n response to the pH d i f f e r e n c e between the cytoplasm and the supporting medium (Ingledew, 1982). The a b i l i t y of weak acids to permeate membranes req u i r e s that the a c i d be protonated; t h i s , i n t u r n , w i l l l e a d to a c i d i f i c a t i o n of the c e l l m atrix. Alexander et a l . , supported t h i s p r e d i c t i o n by demonstrating t h a t organic a c i d t o x i c i t y i s d i r e c t l y r e l a t e d to the pKa values of each a c i d (Alexander et a l . , 1987). The source of the i n h i b i t o r y organic compounds was i n v e s t i g a t e d by Schnaitman and Lundgren i n 1965. Using r a d i o i s o t o p e s , i t was shown that the m a j o r i t y of the p y r u v i c a c i d accumulating i n spent media was from C0 2 f i x e d by T. ferrooxidans and subsequently rele a s e d i n t o the media (Schnaitman & Lundgren, 1965). From these observations, the g e n e r a l l y accepted c o n c l u s i o n i s that end product i n h i b i t i o n plays an important r o l e i n auto t r o p h i c a c i d i p h i l i c growth on mineral s u b s t r a t e s . Contrary to the de t r i m e n t a l e f f e c t of a c i d mine drainage, the 6 b e n e f i c i a l uses of a c i d o p h i l e s have been p r a c t i c e d f o r thousands of years. B i o l e a c h i n g , through the o x i d a t i o n of metal sulphides, has been s u c c e s s f u l l y a p p l i e d on an i n d u s t r i a l s c a l e to the recovery of copper and uranium from low grade ores and t a i l i n g s . I t has been reported that 15-20% of commercially produced copper i n the U.S. i s achieved through b i o l e a c h i n g w i t h a d e f i n i t e p o t e n t i a l t o expand to other ores and ore concentrates (Agate & Khinvasara, 1985). Over the l a s t three decades, s e v e r a l a r t i c l e s have been w r i t t e n on b i o l e a c h i n g . B i o l e a c h i n g i n v o l v e s the b a c t e r i a l o x i d a t i o n of i r o n , elemental sulphur and mineral sulphides such as p y r i t e (FeS2) . This process r e s u l t s i n d i r e c t and i n d i r e c t s o l u b i l i z a t i o n of metals t o be commercially e x t r a c t e d . The d i r e c t mechanism i n v o l v e s the d i r e c t b i o l o g i c a l o x i d a t i o n of an i n s o l u b l e d i v a l e n t metal sulphide t o s o l u b l e metal and sulphate. I t g e n e r a l l y b e l i e v e d that d i r e c t attack r e q u i r e s b a c t e r i a l attachment to the mineral since the substrate i s i n s o l u b l e (Bennett & T r i b u t s c h , 1978; Berry & Murr, 1978; Tr i b u t s h , 197 6). Reactions 1 and 2 describe the b a c t e r i a l o x i d a t i o n of elemental sulphur and p y r i t e by the d i r e c t mechanism. (1) 2 S° + 3 0 2 + 2 H20 - — 2 H2S04 (2) 4 FeS 2 + 15 0 2 + 2 H20 2 Fe 2(S0 4) 3 + 2 H2S04 Any b i v a l e n t metal can be s u b s t i t u t e d f o r ferrous sulphide (FeS2) . The i n d i r e c t mechanism of m i c r o b i a l l e a c h i n g i n v o l v e s three steps. (a) In the a c i d environment created by m i c r o b i a l l e a c h i n g , ferrous sulphate i s produced by the chemical o x i d a t i o n of p y r i t e (FeS2) ( r e a c t i o n 3) . (b) B a c t e r i a l o x i d a t i o n of ferrous i r o n to f e r r i c i r o n ( r e a c t i o n 4) (Lacey & Lawson, 1970). (c) Chemical reduction of f e r r i c sulphate through the o x i d a t i o n of a metal sulphide to regenerate ferrous sulphate ( r e a c t i o n 5) (Singer & Stumm, 1970) . (3) 2 FeS 2 + 7 0 2 + 2 H20 2 FeS0 4 + 2 H2S04 (4) 4 FeS0 4 + 0 2 + 2 H2S04 —*~ 2 Fe 2(S0 4) 3 + 2 H20 (5) Fe 3 + + MS Fe 2 + + M2+ + S° The metal sulphide (MS) o x i d i z e d t o a s o l u b l e i o n i c form i n r e a c t i o n 5 f a c i l i t a t e s the establishment of the f e r r o u s - f e r r i c i r o n c y c l e which i s c h a r a c t e r i s t i c of the i n d i r e c t mechanism. B a c t e r i a l o x i d a t i o n of ferrous i r o n t o r e p l e n i s h f e r r i c i r o n l e v e l s ( r e a c t i o n 4) i s c r i t i c a l f o r the maintenance of t h i s c y c l e . I f r e a c t i o n 5 i s not allowed to occur, then f e r r i c sulphate w i l l r e a d i l y react w i t h water t o form f e r r i c hydroxide and s u l p h u r i c a c i d . Since a l l re a c t i o n s i n the i n d i r e c t mechanism occur i n s o l u t i o n , attachment i s not considered necessary. Though separate systems f o r i r o n and sulphur o x i d a t i o n are e v i d e n t (Beck & Brown, 1968), they appear to f u n c t i o n c o n c u r r e n t l y ( S i l v e r m a n , 1967) . S u l p h u r i c a c i d i s an end p r o d u c t f o r b o t h r e a c t i o n sys tems. The r e s u l t a n t low pH p l a y s an impor tant r o l e i n b i o l e a c h i n g . A c i d i c pH p r o v i d e s s e l e c t i v e p r e s s u r e f o r the enr ichment o f a c i d o p h i l i c l i t h o t r o p h s and h e t e r o t r o p h s . At a low pH, f e r r o u s i r o n i s s t a b l e and w i l l not a u t o - o x i d i z e t o f e r r i c i r o n , t h e r e f o r e , t h i s impor tant s u b s t r a t e remains a v a i l a b l e to the b a c t e r i a . Under low pH c o n d i t i o n s , a membrane p o t e n t i a l o f a p p r o x i m a t e l y -10 mV between the a c i d i c environment and the n e u t r a l i n t e r n a l pH o f T.ferrooxidans i s g e n e r a t e d (Ingledew & C o b l e y , 1980). T h i s c r e a t e s an e l e c t r o c h e m i c a l p r o t o n a c t i v i t y d i f f e r e n c e o f 256 mV which i s s u f f i c i e n t f o r ATP g e n e r a t i o n , as demonstrated by Ingledew et a l . V a r i o u s parameters e f f e c t b i o l e a c h i n g r a t e s (Lundgren & S i l v e r , 1980) . I n c r e a s i n g t e m p e r a t u r e , w i t h i n a range ( 2 5 - 4 5 ° C ) , r e s u l t s i n not o n l y an i n c r e a s e i n c h e m i c a l r e a c t i o n r a t e s , but a l s o an i n c r e a s e i n b a c t e r i a l metabo l i sm. The pH o f 1 .0 -2 .5 i s c o n s i d e r e d o p t i m a l f o r f e r r o u s i r o n o x i d a t i o n . As w i t h most m i c r o o r g a n i s m s , a c i d o p h i l e s r e q u i r e s t a n d a r d m i n e r a l s a l t s . In many c a s e s , depending on the ore c o m p o s i t i o n , i t may be o n l y n e c e s s a r y t o supplement the l e a c h i n g media w i t h ammonia and phosphate . C0 2 and 0 2 a v a i l a b i l i t y i s d i r e c t l y r e l a t e d t o the l e a c h i n g e f f i c i e n c y . Both gases may become l i m i t i n g i f p u l p 9 d e n s i t i e s are too high. The p a r t i c l e s i z e i s a l s o an important parameter; decreased p a r t i c l e s i z e causes an increase i n surface area r e s u l t i n g i n an increased l e a c h i n g e f f i c i e n c y (Torma, 1977). Torma and Bosecker addressed the growing i n t e r e s t i n commercially a p p l i e d bio-hydrometallurgy by p r o v i d i n g a systematic review of current techniques i n mineral l e a c h i n g by microorganisms. Various methods of i n d u s t r i a l b i o l e a c h i n g have been developed; a i r l i f t p e r c o l a t o r s , column le a c h i n g , s t a t i o n a r y l e a c h i n g and a g i t a t e d vat l e a c h i n g . They found that f o r best r e s u l t s , the r a t e of oxygen and carbon d i o x i d e mass t r a n s f e r must be at a maximum. The authors s t r e s s that i n order f o r commercial a p p l i c a b i l i t y , an increased understanding of the m i c r o b i a l leach process and the symbiotic growth phenomena i s e s s e n t i a l (Torma & Bosecker, 1982) . One a p p l i c a t i o n of i n d u s t r i a l b i o l e a c h i n g which i s of growing i n t e r e s t i s the d e s u l p h u r i z a t i o n of c o a l as a means of curbing sulphur d i o x i d e and n i t r o g e n oxide emissions. Both emissions i n t e r a c t w i t h water molecules i n the atmosphere to form s u l p h u r i c a c i d and n i t r i c a c i d , hence a c i d r a i n ( L a B a s t i l l e , 1981). P u b l i c concerns of a c i d r a i n has encouraged implementation of the Clean A i r Act i n the U.S. which r e s t r i c t s the sulphur content of combustible c o a l t o 0.7%. Unfortunately, h a l f of the U.S. c o a l reserves i s bituminous i n nature w i t h i t s m a j o r i t y c o n t a i n i n g greater than 1% sulphur ( M i l l s , 1985). This problem i s compounded by the f a c t that 47% of the U.S. energy needs r e s u l t from c o a l combustion and a p r e d i c t e d 5% annual increase i n domestic energy needs i s expected f o r the next s e v e r a l decades (Detz, 1979) . Two common concerns among researchers designing d e s u l p h u r i z a t i o n treatments are economic f e a s i b i l i t y and reduced residence time. Two n o n - b i o l o g i c a l approaches are f l u e gas d e s u l p h u r i z a t i o n and p r e s s u r i z e d f l u i d i z e d bed combustion, however, high costs and excess waste have l i m i t e d t h e i r usefulness (Detz, 1979). The suppression of p y r i t i c sulphur f l o a t a t i o n during c o a l c l e a n i n g i s another approach. Boateng and P h i l l i p s found that by t r e a t i n g the c o a l w i t h kerosene and lime, you encourage c o a l f l o a t a t i o n and discourage p y r i t e f l o a t a t i o n (Boateng & P h i l l i p s , 1977). Therefore, the sulphur c o n t a i n i n g p y r i t e i s removed from the c o a l p r i o r t o combustion, v a r i a t i o n s to t h i s process were attempted by Kempton e l a l . i n 1980 and Townsley et a l . i n 1987. Both researchers showed th a t by c o n d i t i o n i n g the c o a l w i t h T.ferrooxidans, the n a t u r a l f l o a t a b i l i t y of p y r i t e was s i g n i f i c a n t l y reduced. M i c r o b i a l d e s u l p h u r i z a t i o n through b i o l e a c h i n g i s considered the l e a s t expensive route t o precombustion removal of p y r i t e from a broad range of coals (Detz, 1979; Dugan, 1986). Huber et a l . (1984) provided an a n a l y s i s of m i c r o b i a l d e s u l p h u r i z a t i o n of c o a l . Using a mixed flow r e a c t i o n followed by a plug flow r e a c t o r c o n f i g u r a t i o n , o v er 90% o f t h e p y r i t e was removed at an o v e r a l l 9 day r e s i d e n c e t i m e . The k i n e t i c s o f c o a l d e s u l p h u r i z a t i o n was r e c e n t l y r e v i e w e d by Andrews e t a l . (1988). C r i t i c a l 0 2 a v a i l a b i l i t y was met t h r o u g h a i r s p a r g i n g o f s l u r r i e s up t o 50% (Wt/Wt) c o n t a i n i n g 1% s u l p h u r . Workers found t h a t by r e c y c l i n g c e l l c u l t u r e s , a p o p u l a t i o n o f h e t e r o t r o p h s e s t a b l i s h e d t h e m s e l v e s and i n c r e a s e d p r o c e s s r a t e s not o n l y by removing T.ferrooxidans i n h i b i t o r s but a l s o by i n c r e a s i n g C0 2 l e v e l s such t h a t an exogenous so u r c e o f C0 2 was not n e c e s s a r y . Q u e s t i o n s w h i c h have r e s u l t e d i n h e a t e d debate among r e s e a r c h e r s i n t h i s f i e l d a r e : I s attachment n e c e s s a r y f o r m i n e r a l l e a c h i n g , and i f so, i s t h e adherence s e l e c t i v e and by what mechanism does i t o c c u r ? C l e a r l y , t h e r e i s an advantage t o t h e organism t o be a t t a c h e d t o t h e m i n e r a l s u r f a c e , s i n c e i t i s t h e n p r o x i m a l t o t h e i n s o l u b l e o x i d i z a b l e s u b s t r a t e . E a r l y e v i d e n c e o f T.ferrooxidans d i r e c t i n t e r a c t i o n w i t h s u l p h u r was p r o v i d e d by Voger (Voger & Umbreit, 1941). S c h a e f f e r d e m onstrated t h a t T.thiooxidans t e n a c i o u s l y b i n d s t o s u l p h u r and c o n c l u d e d t h a t attachment i s a p r e r e q u i s i t e f o r s u l p h u r o x i d a t i o n ( S c h a e f f e r & H o l b e r t , 1963). The s t r e n g t h and i r r e v e r s i b i l i t y o f m i n e r a l s u r f a c e b i n d i n g was f u r t h e r demonstrated by B a l d e n s p e r g e r e t a l . i n 1974 w i t h T. thiooxidans and T. denitrificans and by Myerson and K l i n e i n 1983 w i t h T.ferrooxidans. I n c r e a s i n g pH appeared t o have no e f f e c t on T.ferrooxidans d e s o r p t i o n nor d i d repeat washing (Tuovinen et a l . , 1983). M i l d s o n i c a t i o n or treatment w i t h s u r f a c t a n t s were a l s o i n e f f e c t i v e at removing T.ferrooxidans c e l l s bound to c h a l c o p y r i t e (McGorman et a l . , 1969) . T.ferrooxidans appears t o bi n d e x t e n s i v e l y t o mineral surfaces. Wakao et a l . i n 1984 recorded t h a t almost 100% of a T.ferrooxidans c e l l suspension adsorbed to e i t h e r the p y r i t e c r y s t a l s or the w a l l s of the gl a s s tube. McGorman found that over 97% of a c e l l suspension bound to c h a l c o p y r i t e (McGorman et a l . , 1969). Gormely and Duncan reported that 65% of a T.ferrooxidans p o p u l a t i o n a s s o c i a t e d d i r e c t l y w i t h the z i n c sulphide substrate (Gormely & Duncan, 1974). The adherence to mineral surfaces occurs q u i t e r a p i d l y . Myerson and K l i n e observed t h a t over h a l f the c e l l s i n a suspension adsorbed to coa l p a r t i c l e s or glass beads w i t h i n f i v e minutes (Myerson & K l i n e , 1983). Twenty-five percent of a T.ferrooxidans c e l l suspension absorbed w i t h i n f i v e minutes to e i t h e r p y r i t e p a r t i c l e s , f l u o r - a p a t i t e or gl a s s beads (Tuovinen et a l . , 1983). Very s i m i l a r r e s u l t s were recorded by D i S p i r i t o et a l . i n 1983. U n t i l r e c e n t l y , the m a j o r i t y of the evidence demonstrating d i r e c t i n t e r a c t i o n between microorganisms and mineral surfaces has been from e l e c t r o n microscope stud i e s (Lundgren & Tano, 1978; Tr i b u t s c h , 1976; Wakao et a l . , 1984; Bennett & T r i b u t s c h , 1978; Gromely et a l . , 1974; Murr & Berry, 1976; Wiess, 1973; Duncan & Drummond, 1973; H i l t u n e n , 1981; K e l l e r & Murr, 1982; B r i e r l e y e t a l . , 1973). S c a n n i n g e l e c t r o n m i c r o s c o p e (SEM) s t u d i e s ( B e r r y & Murr, 1978; K e l l y e t a l . , 1979) suggest t h a t T.ferrooxidans attachment o c c u r s s e l e c t i v e l y t o s u l p h i d e phases r a t h e r t h a n t o the s i l i c a t e m a t r i x o f p r e p a r e d m i n e r a l samples. Bennett & T r i b u t s c h p e r f o r m e d SEM s t u d i e s on p y r i t e c r y s t a l s w hich were p l a c e d i n a T.ferrooxidans c u l t u r e f o r 2 y e a r s . They obse r v e d e t c h e d p i t s i n t h e shape o f b a c t e r i a l c e l l s w hich were o r i e n t e d i n a " p e a r l - l i k e " s t r i n g . These l i n e s o f b a c t e r i a l c o r r o s i o n were o f t e n i n t e r s e c t e d a t r i g h t a n g l e s by o t h e r c o r r o s i o n l i n e s , t h e r e f o r e r e s e m b l i n g th e c u b i c s t r u c t u r e o f c r y s t a l l i n e p y r i t e . B a g d i g i a n and Myerson (198 6) demonstrated t h a t T.ferrooxidans s e l e c t i v e l y a dsorb t o exposed p y r i t e c r y s t a l s d i s p e r s e d t h r o u g h o u t t h e o r g a n i c c o a l m a t r i x . F u r t h e r m o r e , p r e f e r e n t i a l attachment was obs e r v e d a l o n g f r a c t u r e l i n e s and d i s l o c a t i o n p o i n t s o f t h e p y r i t e c r y s t a l . S e l e c t i v e a d s o r p t i o n t o p y r i t e c r y s t a l s was a l s o d emonstrated by Kempton e t a l . (1980) and Townsley e t a l . (1987) as a means o f d e s u l p h u r i z a t i o n o f c o a l ( p r e v i o u s l y d i s c u s s e d ) . A c c o r d i n g t o Weertman and Weertman (1964), s t r e s s and s t r a i n e n e r g i e s a r e s t o r e d i n d i s l o c a t i o n s i t e s i n t h e form o f c r y s t a l f o r m a t i o n s and l a t t i c e v a c a n c i e s . W i t h t h i s i n mind, Andrews found t h a t d i f f u s i o n o f s u l p h u r t o d i s l o c a t i o n s i t e s o f p y r i t e c r y s t a l s was s i g n i f i c a n t and as such, i t would be advantageous f o r t h e b a c t e r i a t o adsorb t o t h e e n e r g e t i c a l l y r i c h s u l p h u r w h i c h accumulates a t th e d i s l o c a t i o n s i t e (Andrews et a l . , 1988) . S t u d i e s i n v o l v i n g s u r f a c t a n t s and w e t t i n g agents have a l s o produced ev idence l i n k i n g c e l l - s u r f a c e i n t e r a c t i o n w i t h i n c r e a s e d r a t e s o f i r o n and s u l p h u r o x i d a t i o n . I t i s l i k e l y t h a t these substances e f f e c t the i n t e r a c t i o n o f b a c t e r i a and the m i n e r a l s u r f a c e . A c c o r d i n g t o Kingma et a l . (1979), Tween reduces the energy r e q u i r e d by the b a c t e r i u m t o overcome the dynamic s u r f a c e t e n s i o n between the s u b s t r a t e and the l i q u i d medium, thus f a c i l i t a t i n g c o n t a c t between the b a c t e r i u m and the s u b s t r a t u m . S t a r k e y et a l . observed an i n c r e a s e d s u l p h u r o x i d a t i o n i n shake f l a s k c u l t u r e s when F e r g i t o l - 0 8 or Tween 80 was added (S tarkey , 1956) . An enhanced r a t e and ex tent o f l e a c h i n g was r e c o r d e d by Duncan et a l . (1964) when e i t h e r Tween, 20, 40, 60 or 80, or T r i t o n X-100 was added t o T. ferrooxidans c u l t u r e s growing on c h a l c o p y r i t e . Moreover , they found t h a t the Tween t o ore r a t i o was more i m p o r t a n t , w i t h r e g a r d s to l e a c h i n g r a t e s , than the Tween t o media r a t i o , s u g g e s t i n g t h a t Tween a c t s to i n c r e a s e the c o n t a c t between the m i n e r a l s u r f a c e and the m i c r o o r g a n i s m s . A s i d e from i n c r e a s i n g s e l e c t i v e c o n t a c t between the b a c t e r i u m and the subs tra tum (Duncan et a l . , 1973), Tween was found to i n c r e a s e the r a t e o f c o n t a c t by e l i m i n a t i n g the l a g p e r i o d p r i o r to p y r i t e o x i d a t i o n (Roy et a l . , 1981) . Torma et a l . (1976), r e f u t e d these f i n d i n g by r e c o r d i n g a reduced c h a l c o p y r i t e o x i d a t i o n by T.ferrooxidans i n the presence o f s u r f a c e a c t i v e agents (Tween 20, 40, 60 and 80) and o r g a n i c s o l v e n t s . They e x p l a i n e d t h a t the surface a c t i v e agents reduced the surface t e n s i o n and oxygen concent r a t i o n of the medium r e s u l t i n g i n a decreased o x i d a t i o n r a t e . There i s evidence suggesting t h a t surface a c t i v e agents may i n f a c t be endogenously produced w i t h i n b i o l e a c h i n g systems. Wakao et a l . (1983), proposed that the enhanced p y r i t e o x i d a t i o n observed i n T.thiooxidans and T. ferrooxidans mixed c u l t u r e s was due to a s u r f a c t a n t , i d e n t i f i e d as p h o s p h o t i d y l i n o s i t o l (Schaeffer & Umbreit, 1963) secreted by T.thiooxidans. This s u r f a c t a n t i s b e l i e v e d t o reduce the T.ferrooxidans adherence, t h e r e f o r e a l l o w i n g i t to o x i d i z e i r o n more r e a d i l y i n suspension. However, i n 1961, Jones and Starkey i d e n t i f i e d a s u r f a c t a n t produced by T.thiooxidans which enhanced sulphur o x i d a t i o n by wetting the substrate and i n c r e a s i n g b a c t e r i a l adherence. Evidence r e l a t i n g the importance of attachment and substrate o x i d a t i o n was r e c e n t l y reviewed by Espejo et a l . (1987) and Yeh et a l . (1987). T.ferrooxidans growth on sulphur p r i l l s was found to be s e l e c t i v e and s a t u r a b l e . Radioisotope stud i e s had shown that only the attached c e l l s were a c t i v e l y d i v i d i n g . The unattached c e l l s l o s t v i a b i l i t y w i t h a h a l f - l i f e of 3.5 days (Espejo et a l . , 1987). Yeh et a l . (1987) used e p i f luorescence microscopy t o measure mineral adherence and metabolic a c t i v i t y . Through d i f f e r e n t i a l fluorescence, they concluded that the c e l l s most a c t i v e l y i n v o l v e d i n the l e a c h i n g process are those attached to the mineral surface. In general, attachment during b a c t e r i a l l e a c h i n g i s advantageous f o r the microorganism. In a d d i t i o n to clo s e p r o x i m i t y of subst r a t e , attached organisms show greater r e s i s t a n c e t o pH changes, temperature changes and end product t o x i c i t y (Karamanev & Nikolov, 1988). There have been few complete stud i e s on the adherence k i n e t i c s of a c i d o p h i l e s . Adsorption to mineral surfaces appears to occur independently of pH, u n l i k e adsorption to glass beads which increased w i t h decreasing pH (Takakuwa et a l . , 197 9; Tuovinen et a l . , 1983; D i S p i r i t o et a l . , 1983). C e l l v i a b i l i t y was not a requirement f o r adherence (Beck, 1967), however, Tuovinen et a l . observed a s l i g h t o v e r a l l r eduction i n adherence w i t h u l t r a v i o l e t l i g h t t r e a t e d c e l l s . Adsorption experiments by Badigian et a l . (1986) suggests t h a t adsorption occurs through a combination of f i r s t order, r e v e r s i b l e and second order i r r e v e r s i b l e k i n e t i c s . Another a c i d o p h i l e of i n t e r e s t concerning mineral surface attachment i s the t h e r m o p h i l i c l i t h o t r o p h , Sulfolobus. Weiss (1973), through SEM s t u d i e s , observed Sulfolobus adhering t o sulphur p a r t i c l e s v i a a p i l u s and c o r r e l a t e d attachment w i t h the number and lengths of the surface p i l i . By in c u b a t i n g Sulfolobus i n sulphur, S h i w e r s et a l . (1973) saw a gradual increase i n the number of attached organisms. They a l s o c o r r e l a t e d increased sulphur o x i d a t i o n w i t h increased attachment. A comprehensive study of Sulfolobus attachment to c h a l c o p y r i t e drew the f o l l o w i n g conclusions (Berry & Murr, 1976): 1) . Strong support that d i r e c t contact i s necessary for chalcopyr i te ox idat ion . 2) . Evidence of p r e f e r e n t i a l attachment by Sulfolobus to mineral surfaces . 3) . Attachment corre la ted with i ron and copper d i s s o l u t i o n . 4) . Increased surface area corresponds with an increase i n c e l l attachment. 5) . Evidence of "biomatter" production at b a c t e r i a l attachment s i t e s . The molecular mechanism by which acidophi les attach to mineral surfaces i s poorly understood. Takakuwa et a l . (1979) postulated the involvement of sul fhydryl-groups i n T.thiooxidans adherence to sulphur p a r t i c l e s since su l fhydry l -b ind ing reagents blocked attachment. This view i s disputed by Bryant et a l . (1984) who i d e n t i f i e d a glycocalyx-mediated adsorption of T.albertis to sulphur d i s c s . LPS extract ion caused reduced adherence to p y r i t e , sulphur or f l o u r o - a p a t i t e , though, i t i s l i k e l y that other surface components would also be removed during the extract ion (DeSpirito et a l . , 1983). In addi t ion to f l a g e l l a , surface appendages have been reported to ex is t on Thiobacillus spp. (Gonzalez et a l . , 1987; D i S p i r i t o et a l . , 1981), however, the function of the appendages has not been determined. 18 Adherence M a r s h a l l et a l . (1980) d i v i d e s adherence i n t o three separate c a t e g o r i e s : A) S p e c i f i c permanent adhesion. This may a l s o be c a l l e d a c t i v e adherence i n which the b a c t e r i a attaches to a surface by means of complementary surface s t r u c t u r e s . The m a j o r i t y of t h i s form of adhesion has been described i n b a c t e r i a - e p i t h e l i u m i n t e r a c t i o n s . B) N o n - s p e c i f i c permanent adhesion, i s found i n microorganisms which adhere to a v a r i e t y of surfaces i n t h e i r n a t u r a l environment. The surfaces o f t e n d i f f e r c o n s i d e r a b l y suggesting that a n o n - s p e c i f i c mechanism such as polymer-bridging may be i n v o l v e d . C) Temporary adhesion, i s observed i n g l i d i n g b a c t e r i a i n which contact to a s o l i d surface i s necessary f o r movement. Adherence i s a very complex process and has been the subject of numerous s t u d i e s . To understand the mechanism of adherence, the researcher must consider charge, w e t t a b i l i t y , and adsorbed medium components ( e l e c t r o l y t e s and p r o t e i n s ) . Moreover, the observer must not assume th a t the c e l l surface physiology of the b a c t e r i a remains s t a t i c throughout the adherence r e a c t i o n ; i n t e r a c t i o n w i t h an i n t e r f a c e can induce changes i n b a c t e r i a l surfaces (Fl e t c h e r et a l . , 1980). Adhesion has been described to proceed from a r e v e r s i b l e s t a t e i n v o l v i n g n o n - s p e c i f i c bonding to an i r r e v e r s i b l e permanent i n t e r a c t i o n (Marshall et a l . , 1971). Doyle et a l . (1982) p o s t u l a t e d that permanent i r r e v e r s i b l e adherence i s achieved through the a d d i t i v e e f f e c t of n o n - s p e c i f i c i n t e r a c t i o n s a f t e r i n i t i a l contact. The complexity of b a c t e r i a l adherence i s e x e m p l i f i e d by Doyle's i n t e r p r e t a t i o n of p o s i t i v e c o o p e r a t i v i t y i n Streptococcus sanguis b i n d i n g to h y d r o x y l a p a t i t e (Nesbitt et a l . , 1982), i n which the adsorption of one c e l l encouraged the b i n d i n g of adjacent c e l l s . He suggests that once a c e l l binds, p e l l i c l e p r o t e i n s adjacent may change conformation c r e a t i n g new receptors and encouraging the adherence of other c e l l s . The molecular mechanism of adherence has been d i v i d e d i n t o four forms: (1) Chemical bonds which in c l u d e e l e c t r o s t a t i c , covalent and hydrogen bonds (Marshall, 1980). (2) Dipole i n t e r a c t i o n s such as d i p o l e - d i p o l e , dipole-induced d i p o l e and i o n - d i p o l e i n t e r a c t i o n s (Heckels et a l . , 1976; Stotzky, 1980). (3) Hydrophobic bonding (Doyle et a l . , 1982; Beachey, 1981). (4) L e c t i n - l i k e bonding (Duguid et a l . , 1966; C i s a r et a l . , 1983). 20 The f i r s t three types of adherence are g e n e r a l i z e d as being non-s p e c i f i c i n nature. Whereas l e c t i n - l i k e bonding i s a h i g h l y s p e c i f i c form of adherence. Chemical bonding, d i p o l e - i n t e r a c t i o n s and hydrophobic bonding are considered physicochemical adherence mechanisms and are discussed below. Using w e l l - d e f i n e d , n o n - l i v i n g systems, Rutte r & Vincent (1984), attempted to r e l a t e s t u d i e s of p a r t i c l e adhesion t o the understanding of m i c r o b i a l adherence to surfaces. They recognized a l i m i t a t i o n of such an approach was that surface charges are not s t a t i c and are often adaptive. A t r u e physicochemical e q u i l i b r i u m may not be reached because m i c r o b i a l attachment does not occur i n a c l o s e d system; i n t e r n a l or surface s t r u c t u r a l changes to the microorganism may occur during the adherence process. They s t r e s s that i n order t o understand the adhesive j u n c t i o n between a c e l l and substratum, a l l i n t e r a c t i o n s i n v o l v i n g the surfaces and the environment must be considered. In order f o r surfaces of s i m i l a r charge to come i n t o contact w i t h each other, a l a r g e free energy b a r r i e r must be overcome. They define t h i s free energy based on van der Waals forces and the overlap of the e l e c t r i c a l double l a y e r a s s o c i a t e d w i t h charged groups on each surface. This energy b a r r i e r i s i n f l u e n c e d by the e l e c t r o l y t e c o n c e n t r a t i o n i n the surrounding medium. As the e l e c t r o l y t e c oncentration i n c r e a s e s , the energy b a r r i e r decreases u n t i l a net a t t r a c t i o n occurs between surfaces. In cases i n v o l v i n g surfaces of opposite charge, the net e f f e c t i s a strong a t t r a c t i o n which i s only m i l d l y decreased w i t h increased e l e c t r o l y t e c o n c e n t r a t i o n . The p o i n t s discussed i n the preceding paragraph address long-range forces necessary to b r i n g two surfaces i n t o c l o s e p r o x i m i t y . However, short-range f o r c e s , i n the form of d i p o l e i n t e r a c t i o n s and hydrogen bonding, are necessary to achieve adhesion between the two surfaces. In the case of two h y d r o p h i l i c surfaces, there i s a net increase i n energy r e q u i r e d to d i s p l a c e the water molecules, r e s u l t i n g i n short-range r e p u l s i o n . The opposite e f f e c t occurs when two hydrophobic surfaces i n t e r a c t due t o a net decrease i n energy r e q u i r e d f o r water displacement. Macromolecules which l a y e r the surfaces of micro-organisms, can e f f e c t m i c r o b i a l adherence: Macromolecules, shed i n t o the environment, can le a d to increased s o l u t i o n v i s c o s i t y and polymer bridge formation. The l a t t e r occurs when the macromolecule co-adsorbs onto two separate surfaces. Bridge formation i s oft e n enhanced by d i v a l e n t c a t i o n s which l i n k polymers through a c i d i c groups. As more p a r t i c l e s of s i m i l a r surface charge adsorb t o a surface, there are l a t e r a l i n t e r a c t i o n s which must be considered. The 22 nature of these forces are dependant on the e l e c t r o l y t e c o n c e n t r a t i o n : In low e l e c t r o l y t e concentrations, adsorbing p a r t i c l e s tend to repulse one another. However, under high e l e c t r o l y t e concentrations, the l a t e r a l forces are a t t r a c t i v e . At intermediate e l e c t r o l y t e concentrations a compromise between a t t r a c t i v e and r e p u l s i v e forces i s reached. This i s d e f i n e d as the second minimum i n which p a r t i c l e s are maintained at some distance from the substratum surface by the opposing f o r c e s . Second minimum adherence i s of low a f f i n i t y and r e v e r s i b l e . High a f f i n i t y , i r r e v e r s i b l e adherence i s achieved only i f the necessary f r e e energy to overcome the primary and l a t e r a l forces i n a v a i l a b l e . Rutter and Vincent summarize by acknowledging that long-range and short-range forces w i l l not completely d i c t a t e a p a r t i c l e s adhesive behaviour. These forces f u n c t i o n t o r e v e r s i b l y h o l d a p a r t i c l e adjacent t o a surface, t h e r e f o r e a l l o w i n g the opportunity f o r i r r e v e r s i b l e adsorption. The p a r t i c l e must withstand hydrodynamic and shear forces from bulk medium flow. U l t i m a t e l y , the p a r t i c l e / s u r f a c e s p e c i f i c i t i e s w i l l draw the f i n a l l i n k between a c e l l and the substratum. By studying polymer adsorption t o surfaces, Robb et a l . (1984) p r e d i c t s the i n f l u e n c e of e x o c e l l u l a r polymers on b a c t e r i a adherence to surfaces. Polymer adsorption to surfaces appears to be i r r e v e r s i b l e . This i s p r i m a r i l y due to the formation of a 23 l a r g e number of r e l a t i v e l y weak bonds between the polymer and the substratum. Negatively charged b a c t e r i a adhering v i a polymers t o ne g a t i v e l y charged surface, i l l u s t r a t e s the streng t h of polymer adsorption. Polymer a c t i v e s i t e s r e s p o n s i b l e f o r adsorption i n c l u d e p o s i t i v e and n e g a t i v e l y charged regions of e x o c e l l u l a r polysaccharides and p r o t e i n s . Since polymers are often h i g h l y charged, the i o n i c s trength of the media i s an important f a c t o r concerning polymer-surface adsorption. The a f f i n i t y of a c e l l surface polymers f o r the solvent i s important i n preventing the c e l l from aggregating i n the s o l u t i o n and maintaining the polymer i n an extended s t a t e , thus, a v a i l a b l e f o r substratum i n t e r a c t i o n . Polymer extension from the c e l l surface i s oft e n s u f f i c i e n t enough to contain the net negative charge of the c e l l w a l l . As a r e s u l t , the net charge of the c e l l i s a t t r i b u t e d to the polymer i t s e l f . K j e l l e b e r g (1984), through the study of var i o u s environmental f a c t o r s , attempts t o l i n k s t u d i e s on n o n - b i o l o g i c a l adhesion t o n a t u r a l m i c r o b i a l ecology. Growing evidence suggests a c o r r e l a t i o n between b a c t e r i a and microzones of increased n u t r i e n t c o n c e n t r a t i o n . Most organisms adhere to surfaces by means of e x o c e l l u l a r polymers. The composition and qu a n t i t y of polymer has been l i n k e d to environmental c o n d i t i o n s . Rosenberg et a l . (1983a) 24 demonstrated t h a t c e l l s t a r v a t i o n s t r o n g l y decreased the degree of encapsulation of Acinetobacter calcoacetius which i n t u r n increased i t s c e l l surface hydrophobicity. This change i n hydrophobicity markedly e f f e c t e d i t s adhesive p r o p e r t i e s as i l l u s t r a t e d by a reduced adherence to hydrocarbons. I t i s b e l i e v e d that a s u b t l e balance e x i s t s between c e l l surface components which encourage and discourage adherence. The presence of LPS reduced c e l l surface hydrophobicity of Salmonella typhimurium and, as a r e s u l t , decreased i t s adherence to the a i r -water i n t e r f a c e (Hermansson et a l . , 1982). A l t e r n a t i v e l y , the appearance of f i m b r i a e was found to increase c e l l surface hydrophobicity. Adherence s p e c i f i c i t y has been the subject of i n c r e a s i n g debate. For example, Rosenberg et a l . (1983b) demonstrated t h a t the o i l degrading Acinetobacter calcoacetius while adhering t o the o i l -water i n t e r f a c e a l s o r e a d i l y adhered to t o o t h surfaces and e p i t h e l i a l c e l l s . Instead of a s t e r e o - s p e c i f i c component resp o n s i b l e f o r adherence, they argue t h a t these components c o n t r i b u t e to an o v e r a l l hydrophobic c e l l surface which was i n t u r n r e s p o n s i b l e f o r i t s adherence d i v e r s i t y . In 1973, M a r s h a l l and Cruickshank f i r s t drew a c o r r e l a t i o n between c e l l h ydrophobicity and surface adherence. In support of t h i s , Dahlback et a l . (1981) performed hydrophobicity assays on a broad range of b a c t e r i a l i s o l a t e s from both the a i r - w a t e r i n t e r f a c e and subsurface water. Their r e s u l t s suggested that i s o l a t e s w i t h the 25 highest degree of hydrophobicity were a s s o c i a t e d w i t h the a i r -water i n t e r f a c e . Using thermodynamic models, m i c r o b i a l adherence to hydrophobic surfaces i s p r e d i c t a b l y more favourable than adherence to h y d r o p h i l i c surfaces, provided the surface t e n s i o n of the l i q u i d medium i s higher than that of the b a c t e r i a l surface. This i s the case w i t h most n a t u r a l environments and i s supported by experimental data of other workers ( K j e l l e b e r g , 1984) . Various c e l l surface components have been found to act as adhesins. The abundance and composition of these s t r u c t u r e s vary markedly between species and w i t h i n species of microorganisms. The growth c o n d i t i o n s and the p h y s i o l o g i c a l s t a t e of the c e l l can have a s i g n i f i c a n t e f f e c t on the adhesion process. Leech and Heffor (1980) demonstrated an inverse r e l a t i o n s h i p between growth r a t e and S.sanguis d e p o s i t i o n on i n e r t polystyrene l a t e x p a r t i c l e s . Rosan et a l . (1982) f u r t h e r demonstrated t h a t carbohydrate source and growth pH a l s o a f f e c t s S.sanguis adherence. E x t r a c e l l u l a r polymers such as l i p o p o l y s a c c h a r i d e (LPS), of gram negative organisms, and l i p o t e i c h o i c a c i d (LTA) of gram p o s i t i v e organisms can p l a y a c r u c i a l r o l e i n c e l l adherence (Dazzo & Truchet, 1983; Beachey, 1981). LPS mediated adherence i s best demonstrated by the Rhizobium-26 legume symbiosis (Dazzo et a l . , 1982). S p e c i f i c r e v e r s i b l e attachment i s a necessary step towards host c e l l i n f e c t i o n and n i t r o g e n - f i x i n g root module formation. The surface components mediating attachment i n c l u d e a m u l t i v a l e n t l e c t i n ( t r i f o l i i n A) on the root h a i r , and carbohydrate receptors i n the f i b r i l l a r capsule of the Rhizobium c e l l s urface. T r i f o l i i n A l e v e l s on root h a i r s was found t o be regu l a t e d by exogenous n i t r a t e supply (Dazzo & B r i l l , 1978). The l e v e l s of the l e c t i n - b i n d i n g saccharide (quinovosamine) i n Rhizobium LPS i s growth dependent and increases s i g n i f i c a n t l y as c e l l s enter s t a t i o n a r y phase (Hrabak et a l . , 1981). LPS i s h i g h l y d i v e r s e among gram negative organisms. The LPS molecule c o n s i s t s of three d i s t i n c t regions; O-polysaccharides, core polysaccharide and the l i p i d A regi o n . The l i p i d A region i s h i g h l y conserved p o r t i o n of LPS c o n s i s t i n g of phosphorylated glucosamines residues i n the form of o l i g o s a c c h a r i d e s which are attached t o f a t t y a c y l e s t e r s , thus p r o v i d i n g the hydrophobic, membrane bi n d i n g , p o r t i o n of t h i s a m p h i p h i l i c molecule. The core polysaccharide i s al s o h i g h l y conserved and i s c h a r a c t e r i z e d by a l d o h e p t o s e and 2 - k e t o - 3 - d e o x y o c t o n a t e r e s i d u e s . Ketodeoxyoctonate along w i t h s e v e r a l phosphate residues c o n t r i b u t e t o a net negative charge i n t h i s region of the molecule. The O-polysaccharides are polymers of repeating sequences of two t o four mono-saccharides which in c l u d e a wide range of heptoses and pentoses. The h y d r o p h i l i c polysaccharide region of the molecule determines the s e r o t y p i c d i f f e r e n c e s between s t r a i n s of a b a c t e r i a l species (Wicken, 1985). Less s p e c i f i c adherence by surface polymers i s mediated by c e l l surface S l a y e r s or by b a c t e r i a l g l y c o c a l y x . S l a y e r s contain a reg u l a r array of g l y c o p r o t e i n subunits which are a s s o c i a t e d w i t h the c e l l w a l l v i a d i v a l e n t c a t i o n s . The g l y c o c a l y x , or c e l l capsule, i s a h i g h l y hydrated polysaccharide matrix which v a r i e s i n complexity from simple homopolymers to complex heteropolymers. This f i b r o u s matrix may be f l e x i b l e or r i g i d and may be shed by the c e l l or i n t e g r a t e d i n t o the outer membrane. The g l y c o c a l y x i s found i n a v a r i e t y of n a t u r a l ecosystems and i s ofte n l o s t upon i n v i t r o s u b c u l t u r i n g . During the attachment process the gl y c o c a l y x i s thought to overcome e l e c t r o s t a t i c r e p u l s i v e f o r c e s , experienced by the b a c t e r i a , by forming a bridge between the c e l l and the surface. Once contact i s e s t a b l i s h e d , weaker forces c o n t r i b u t e towards an i r r e v e r s i b l e adherent s t a t e (Costerton, 1981) . This view has been disputed by Sutherland (1983), suggesting t h a t the g l y c o c a l y x may not be i n v o l v e d i n i n i t i a l attachment, but plays a greater r o l e i n e s t a b l i s h i n g i r r e v e r s i b l e attachment. Prosthecate b a c t e r i a , such as Caulobacter spp., are c h a r a c t e r i z e d by p o l a r extensions, termed s t a l k s . The d i s t a l p o r t i o n of the b a c t e r i a l s t a l k contains a h o l d f a s t adhesive which i s re s p o n s i b l e f o r adherence t o surfaces. The l i f e c y c l e of Caulobacter i s 28 separated by a swarmer c e l l phase and a surface attached phase. The h o l d f a s t , which i s present during both phases of the dimorphic l i f e c y c l e , provides a good system f o r studying the molecular mechanisms of adhesion. C h a r a c t e r i z a t i o n of Caulobacter adherence i s c u r r e n t l y being i n v e s t i g a t e d (Merker & Smit, 1988) . Fimbriae and p i l i are the most widely s t u d i e d c e l l surface appendages. Fimbriae i s o l a t e d from gram negative organisms are short, s t r a i g h t p r o t e i n appendages, made up of subunits which vary from 15 t o 25 Kd (Jones & Isaacson, 1983) . The amino terminus i s h i g h l y conserved and s t r o n g l y hydrophobic suggesting a p o s s i b l e a s s o c i a t i o n w i t h the outer membrane. They are most prominent i n gram negative b a c t e r i a and have been i m p l i c a t e d i n adhesion and c e l l aggregation. The adherence process has a l s o i n v o l v e d the p i l u s . P i l i , u n l i k e f i m b r i a e , are plasmid-encoded lar g e proteinaceous appendages which f a c i l i t a t e t r a n s f e r of genetic m a t e r i a l between donor and r e c i p i e n t c e l l s . Another c e l l surface s t r u c t u r e i n v o l v e d i n adherence are f i b r i l s . Handley et a l . (1985) has described f i b r i l s on a number of gram p o s i t i v e and gram negative organisms. U n l i k e f i m b r i a e , these r a d i a t i n g s t r u c t u r e s do not have a defined width. Duguid et a l . (1955) f i r s t i d e n t i f i e d the n o n - f l a g e l l a r , p o l a r and p e r i t r i c i o u s , short filamentous appendages i n Escherichia coli. These surface s t r u c t u r e s , designated antigen K-88 and K-99 have been shown to mediate E.coli d i a r r h o e a l disease i n p i g l e t s and c a l v e s , r e s p e c t i v e l y (Orskov et a l . , 1961; Orskov et a l . , 1975) . I t i s b e l i e v e d these antigens play a r o l e i n attachment le a d i n g t o v i r u l e n c e since i n f e c t i v i t y i s i n h i b i t e d by adhesin s p e c i f i c a n t i s e r a or p u r i f i e d f i m b r i a e . In a d d i t i o n , non-f i m b r i a t e d s t r a i n s of E.coli were found to be n o n - v i r u l e n t . Since K88 and K99 were shown to be plasmid encoded, they have been c a t e g o r i z e d as p i l i i n s t e a d of fimbriae (Isaacson, 1984). This phenomenon has been observed i n other gram negative organisms. P i l i - m e d i a t e d attachment of pathogenic Neisseria spp. t o mucosal surfaces has been demonstrated (Stephens, 1984). Of a l l the adherence mechanisms discussed, t h i s i s an example of the most s p e c i a l i z e d form since a s p e c i f i c i n t e r a c t i o n between the p i l i and the receptor molecule on the i n t e s t i n a l e p i t h e l i u m i s necessary f o r attachment. A number of d i f f e r e n t techniques have been employed i n the study of m i c r o b i a l adherence. These methods can be d i v i d e d i n t o two general approaches: A) C h a r a c t e r i z a t i o n of the adherence r e a c t i o n . B) I s o l a t i o n and i d e n t i f i c a t i o n of the adhesin. Adherence r e a c t i o n c h a r a c t e r i z a t i o n can i n v o l v e chemical or enzymatic m o d i f i c a t i o n of the c e l l surface. For example, protein-mediated adherence can be e l i m i n a t e d by t r e a t i n g the c e l l s w i t h a protease (Weerkamp et a l . , 1980). S i m i l a r l y , s i a l i c a c i d residue removal by neuraminidase was shown to e f f e c t 30 Streptococcus sanguis i n t e r a c t i o n w i t h s a l i v a r y p r o t e i n s (McBride et a l . , 1977; Levine et a l . , 1978). Chemical treatment of c e l l s can a l s o markedly e f f e c t adherence. For example, treatment w i t h reducing agents i m p l i e d the involvement of • s u l f h y d r y l groups i n the adherence of Sulfolobus to sulphur (Wiess, 1973). Competitive i n h i b i t i o n s t u d i e s using receptor analogues to c h a r a c t e r i z e adherence i s a common approach. Carbohydrate-s e n s i t i v e adherence has been described i n o r a l b a c t e r i a by Duguid et a l . (1966). Competitive b i n d i n g experiments between s i m i l a r or d i f f e r e n t c e l l types has been a u s e f u l t o o l i n studying adherence. Staat et a l . (1984), used competitive b i n d i n g between r a d i o a c t i v e and non-radioactive c e l l s t o d i s t i n g u i s h s p e c i f i c from n o n - s p e c i f i c adherence. Adsorption isotherms have become a u s e f u l t o o l f o r studying adherence s p e c i f i c i t y and k i n e t i c s . The b i n d i n g isotherm i s obtained by p l o t t i n g the number of f r e e c e l l s (U) versus the number of bound c e l l s (B) at e q u i l i b r i u m i n a given system. Information about the adherence k i n e t i c s can be obtained using the Langmuir equation; U/B = K/N + (1/N)U, where K i s the d i s s o c i a t i o n constant and N i s the maximum number of b i n d i n g s i t e s . By p l o t t i n g U/B verses U, an e s t i m a t i o n of the d i s s o c i a t i o n constant and the a f f i n i t y constant, (1/K from the x i n t e r c e p t ) , can be determined. Though the c o e f f i c i e n t of v a r i a t i o n i s high using these isotherms, some u s e f u l i n f o r m a t i o n regarding c e l l - s u r f a c e i n t e r a c t i o n s can be obtained (Gibbons et a l . , 1976) . The more d i f f i c u l t method of studying adherence i s through the i s o l a t i o n and i d e n t i f i c a t i o n of the c e l l adhesin. A popular approach used by many workers i s the i s o l a t i o n of non-adherent mutants. The c e l l surface p r o t e i n r e s p o n s i b l e f o r adherence can i n some cases, be i d e n t i f i e d by comparing the outer membrane p r o f i l e s of the mutant and the wild-type (Fives-Taylor et a l . , 1985; Gaastra et a l . , 1982). Mutants l a c k i n g a surface adhesin can then be used t o adsorb wild-type a n t i s e r a i n order to p u r i f y monospecific p o l y c l o n a l a n t i s e r a t o the adhesin molecule (Fachon-Kalweit et a l . , 1985; C i s a r et a l . , 1983). The a d h e s i n - s p e c i f i c a n t i s e r a i s u s e f u l i n adhesin l o c a l i z a t i o n s t u d i e s through immunogold techniques and i n adherence i n h i b i t i o n s t u d i e s . Several researchers have addressed the question whether attached b a c t e r i a c o n t r i b u t e t o the s u r v i v a l of f r e e - l i v i n g b a c t e r i a . I t i s g e n e r a l l y accepted t h a t i n a n u t r i e n t depleted aquatic environment, organic substrates tend t o accumulate at l i q u i d i n t e r f a c e s . However, i n n a t u r a l aquatic environments, only a small percent of the t o t a l surface area i s c o l o n i z e d by b a c t e r i a (Hoppe, 1984). K h a i l o v and co-workers (1970) made three observations which could e x p l a i n t h i s environmental enigma: 1) High molecular weight macromolecules which are prevalent 32 i n n a t u r a l aquatic systems and d i s p l a y high degrees of surface a c t i v i t y , are almost e x c l u s i v e l y adsorbed t o surfaces. 2) Attached b a c t e r i a e x h i b i t higher e x t r a c e l l u l a r enzymatic c a p a c i t i e s necessary f o r macromolecule degradation than t h e i r f r e e - l i v i n g counterparts. 3) Attached b a c t e r i a do not immediately consume t h e i r products r e s u l t i n g from macromolecular degradation. They i n t e r p r e t these f i n d i n g s t o mean that attached b a c t e r i a b e n e f i t the f r e e - l i v i n g b a c t e r i a by degrading the high molecular weight macromolecules and make a v a i l a b l e s o l u b l e organic s u b s t r a t e s . I w i l l now review the current methods of measuring m i c r o b i a l adherence t o mineral surfaces (Van Es & Meyer-Reil, 1982). Most i n v e s t i g a t o r s have measured c e l l adherence i n d i r e c t l y and determined the concent r a t i o n of unbound c e l l s by c o r r e l a t i n g c e l l numbers wi t h v a r i o u s biomass or metabolic i n d i c a t o r s . Examples of biomass measurement i n d i c a t o r s are: 1) P r o t e i n concentration (Myerson et a l . , 1983; D i S p i r i t o et a l . , 1983). 2) O p t i c a l d e n s i t y measurements at 660 nm (Kakakuwa et a l . , 1979) . 3 3 3) Organic n i t r o g e n concentration (Gormely et a l . , 1974). 4) Adenosine triphosphate (ATP) content (Tuovinen & Sormuneu, 1979). B i o a c t i v i t y i n d i c a t o r s used to measure adherence are: 1) Radioactive carbon f i x a t i o n ( B r i e r l e y , 1977). 2) Radioactive phosphate i n c o r p o r a t i o n (McCready & L e G a l l a i s 1984). The major drawback to i n d i r e c t techniques i s t h a t one cannot d i s t i n g u i s h the b i n d i n g of one c e l l type from another. In a d d i t i o n , microscopic mineral f i n e s , which i n t e r f e r e w i t h many biomass assays, are d i f f i c u l t t o remove from unbound c e l l s l e f t i n suspension. P r o t e i n and n i t r o g e n c o n c e n t r a t i o n assays r e s t r i c t the p o s s i b l e uses of c e r t a i n p r o t e i n or n i t r o g e n -c o n t a i n i n g adherence i n h i b i t o r s . R adioactive isotope s t u d i e s have problems of decay quenching during s c i n t i l l a t i o n counting. ATP assay techniques have encountered d i f f i c u l t i e s w i t h ATP e x t r a c t i o n and measurement i n the presence of high l e v e l s of i r o n compounds and heavy metals i n the samples. The i n t r o d u c t i o n of f l u o r e s c e n t - a n t i b o d y (FA) techniques allowed d i r e c t observation of bound c e l l s (Apel et a l . , 1976; Muyzer et a l . , 1987). In a d d i t i o n , f l u o r e s c e n t - a n t i b o d y microscopy can i d e n t i f y s p e c i f i c c e l l s t r a i n s , p r o v i d i n g the antibody used i s s p e c i f i c . The FAINT technique (Baker & M i l l s , 1982), which i s a modified v e r s i o n of the FA technique, allows the measurement of a c t i v e l y growing c e l l s through the b i o l o g i c a l r e d u c t i o n of I N T ( 2 - ( p - i o d o p h e n y l ) - 3 - ( p - n i t r o p h e n y l ) - 5 phenyl t e t r a z o l i u m c h l o r i d e ) to a pigmented formazan compound which accumulates i n the c e l l . This type of p h y s i o l o g i c a l i n f o r m a t i o n can a l s o be obtained through e p i f l u o r e s c e n c e microscopy (EFM) using a c r i d i n e orange (Yeh et a l . , 1987) which, depending on the a c t i v i t y of the c e l l , w i l l f l u o r e s c e d i f f e r e n t c o l o u r s . However, c o n f l i c t i n g r e s u l t s have been experienced by the l a t t e r two techniques; both authors a t t r i b u t e d these i n c o n s i s t e n c i e s to the unknown e f f e c t s of low pH. Though these d i r e c t microscopic techniques can be i n f o r m a t i v e , FAINT and EFM are labour i n t e n s i v e . In a d d i t i o n , background fluorescence through n o n - s p e c i f i c adsorption by the antibody t o the mineral surface, can i n t e r f e r e w i t h adherence e s t i m a t i o n s . In l i g h t of the drawback discussed regarding current adherence assays, I have developed a n o n - i s o t o p i c , s p e c i f i c , s e n s i t i v e approach to measuring m i c r o b i a l adherence to mineral surfaces. Growing evidence supports the importance of substrate attachment i n i n d u s t r i a l a p p l i c a t i o n s of a c i d o p h i l i c microorganisms; however, a thorough a n a l y s i s of a c i d o p h i l e attachment mechanisms i s l a c k i n g . Given the importance of h e t e r o t r o p h i c a c i d o p h i l e s and t h e i r symbiotic r e l a t i o n s h i p w i t h a u t o t r o p h i c a c i d o p h i l e s , I have l i m i t e d my study p r i m a r i l y to the organism, Acidiphilium 35 cryptum. The purpose of t h i s report then i s to provide a comprehensive study of h e t e r o t r o p h i c a c i d o p h i l e adherence t o mineral surfaces by c h a r a c t e r i z i n g the adherence r e a c t i o n and studying adherence-defective mutants. 36 MATERIALS AND METHODS Bacteria The b a c t e r i a l s t r a i n s used i n t h i s study were A.cryptum, ATCC 33463, and T.ferrooxidans, ATCC 23270. Both c u l t u r e s were obtained from American Type Culture C o l l e c t i o n . Media and stock maintenance A.cryptum was c u l t i v a t e d on a medium c o n t a i n i n g the f o l l o w i n g per l i t r e : (NH 4) 2S0 4, 2 gm; KC1, 0.1 gm; K2HP04, 0.25 gm; MgS04-7H20, 0.25 gm; Ca (N03) 2-4H20, 0.01 gm; dehydrated yeast e x t r a c t (Difco l a b o r a t o r i e s , Detroit,Michigan) , 0.1 gm; D-dextrose, 1.0 gm. This complete media w i l l be r e f e r r e d to as MSGYE medium f o r the re s t of the t e x t . Media without the organic i n g r e d i e n t s was designated b a s a l mineral s a l t s . The ba s a l mineral s a l t s was pH adjusted t o 3.2 wi t h 1 N H2S04. To prepare MSGYE media, double strength b a s a l mineral s a l t s (pH 3.2) and double stre n g t h glucose-yeast e x t r a c t s o l u t i o n s were autoclaved separately at 15 l b / i n 2 f o r 20 min and then a s e p t i c a l l y mixed a f t e r c o o l i n g . For s o l i d media, 15 g m / l i t r e of agar was added t o the glucose-yeast e x t r a c t s o l u t i o n p r i o r to au t o c l a v i n g . Small, white, convex, round c o l o n i e s could be detected w i t h i n three days of growth at 30° C. A.cryptum was stor e d on MSGYE agar s l a n t s at 4° C f o r one month periods between t r a n s f e r s . Stock c u l t u r e s of A.cryptum were maintained at -70° C i n a ba s a l mineral s a l t s s o l u t i o n c o n t a i n i n g 10% g l y c e r o l (pH 3.2). Late log-phase growing c e l l s were harvested from a 3 day c u l t u r e incubated at 30° C. P l a t e -grown c e l l s were ge n t l y removed from the agar surface and washed once i n 1/2 concentration b a s a l mineral s a l t s (1/2 MS), pH 3.2 which w i l l be r e f e r r e d to as adherence b u f f e r . T.ferrooxidans was grown and maintained on 100:10 s o l i d media as described by Schrader and Holmes (1988) . E x p o n e n t i a l l y growing c e l l s were harvested from a 7 day c u l t u r e grown at 30° C and washed once i n adherence b u f f e r . C e l l suspension concentrations were determined using a Petroff-Hausser counting chamber and phase-contrast microscopy. Buffers The adherence b u f f e r c o n s i s t e d of a 1/2 d i l u t i o n of the b a s a l mineral s a l t s which was adjusted to pH 3.2 w i t h 1 N H2S04. For i n h i b i t i o n assays, the v a r i o u s p u t a t i v e i n h i b i t o r s were added to the adherence b u f f e r . Ore ELISA b u f f e r s , PBS/Tween and PBS 1% BSA, were each pH adjusted to 5.0 w i t h 1 N HCl. Bio-Rad Adherence Assay A known number of c e l l s were resuspended i n adherence b u f f e r . 1.5 ml of the c e l l suspension was added to microfuge tubes c o n t a i n i n g pre-measured amounts of mineral ore. This mixture was a g i t a t e d through continuous i n v e r s i o n by the Labquake (L a b i n d u s t r i e s , C a l i f . ) at 20° C f o r the d u r a t i o n of the i n c u b a t i o n p e r i o d . The ore was removed from s o l u t i o n by low 38 speed c e n t r i f u g a t i o n (5800xg) f o r 20 seconds. To c o n t r o l f o r f a l s e p o s i t i v e c e l l l o s s during t h i s step, "spun" and "non-spun" c e l l suspension c o n t r o l s were performed. A 0.9 ml volume of the supernatant, c o n t a i n i n g the unbound c e l l s l e f t i n suspension, was removed to a new microfuge tube. The unbound c e l l s were harvested by high speed c e n t r i f u g a t i o n (13, OOOxg) f o r 10 min. The supernatant was discarded and the c e l l p e l l e t was resuspended i n d i s t i l l e d water. The c e l l s were then t r a n s f e r r e d t o gl a s s tubes and b o i l e d f o r 10 min to l y s e the c e l l s . When the tubes were c o o l , 0.2 ml of Bio-Rad p r o t e i n assay concentrate (Bio-Rad L a b o r a t o r i e s , Richmond, CA.) was added to each tube and p r o t e i n c oncentration was measured at A 5 g s. In order t o c a l c u l a t e adherence, the f o l l o w i n g equation was used: % Adherence = [ 1 - ( t e s t / c e l l spun c o n t r o l )] x 100 The number of c e l l s bound was determined by m u l t i p l y i n g the % adherence by the number of c e l l s o r i g i n a l l y added to the assay tube. Ore ELISA Adherence Assay This assay procedure i s a modified v e r s i o n of the Enzyme-Linked Immunoadsorbent Assay (ELISA), by E n g v a l l (1972). To prevent c e l l adsorption t o p r e p a r a t i v e m a t e r i a l s , a l l glassware was s i l i c o n i z e d w i t h Surfa S i l (Pierce Chem. Co., Rockford, I I . ) . M i n e r a l ore samples were provided by B r i t i s h Columbia Research 39 I n s t , and are d e s c r i b e d i n Table I. The ore samples were washed, des i c c a t e d and f i n e l y ground to an average p a r t i c l e s i z e of 1.5 yum. A coat of s i l i c o n e - b a s e d glue (Dow Corning corp., Midland, MI.) was a p p l i e d t o each w e l l of a fl a t - b o t t o m m i c r o t i t e r p l a t e (Gibco, Canada I n c . ) . Each w e l l was then coated w i t h prepared mineral ore and allowed t o dry overnight (Fig. 5) . Unbound ore was removed and the p l a t e was thoroughly r i n s e d w i t h d i s t i l l e d water t o remove l o o s e l y bound p a r t i c l e s . The p l a t e s were then d r i e d at 37° C f o r one hour. A known number of c e l l s were resuspended i n adherence b u f f e r . 300 yul of c e l l suspension was added to each w e l l and incubated on a r o t a r y shaker (100 rpm) at 20° C. A f t e r the d e s i r e d p e r i o d of Table I. T i t l e and d e s c r i p t i o n of mineral ore. Source Geological constituent Newmont mine chalcopyrite/pyrite Gibraltar mine chalcopyrite/pyrite Empire mine pyrite Cambell Red lake pyrite/arsenopyrite 40 i n c u b a t i o n , the unbound c e l l s were removed and each w e l l was r i n s e d twice w i t h adherence b u f f e r . 400 /AI of b l o c k i n g s o l u t i o n (PBS/1% BSA, pH 5.0) was added to each w e l l and incubated f o r one hour at 37° C. A f t e r the b l o c k i n g s o l u t i o n was removed, the p l a t e was r i n s e d twice w i t h washing b u f f e r (PBS/Tween, pH 5.0). The primary antibody was d i l u t e d i n PBS/1% BSA (pH 5.0) and 300 >*1 was added to each w e l l and incubated overnight at 4° C. The primary antibody was removed and the p l a t e was washed 3 times wi t h washing b u f f e r (the l a s t wash was allowed to s i t f o r 10 min). The secondary antibody s o l u t i o n contained a l k a l i n e phosphatase conjugated goat a n t i - r a b b i t IgG (Helix B i o t e c h l t d . ) d i l u t e d i n PBS/1% BSA (pH 5.0). 300 >»1 of secondary antibody s o l u t i o n was added to each w e l l and incubated at 37° C f o r one hour. The secondary antibody was removed and the p l a t e s were washed 3 times w i t h washing b u f f e r (the l a s t wash was again allowed t o s i t f o r 10 min). The p l a t e s were developed by adding 300 /AI of a l k a l i n e phosphatase substrate (Sigma Chemical Co., St.Louis, Mo.) to each w e l l and in c u b a t i n g at 37° C i n the dark. Upon colour development, 100 ,M1 of spent a l k a l i n e phosphatase substrate was removed t o a second m i c r o t i t e r p l a t e f o r adsorbance reading at 405 nm. In order t o c a l i b r a t e the A 4 0 5 values to b a c t e r i a per m i l l i l i t r e , s p e c i f i c a c t i v i t y measurements were performed f o r each assay run. The antibody r e a c t i v i t y was determined by measuring the A 4 0 5 values of known c e l l c o ncentrations, i n 1 ml volumes, under i d e n t i c a l c o n d i t i o n s as the Ore ELISA assay. This assay was performed i n microfuge tubes t o allow the c e n t r i f u g a t i o n of the c e l l s between each assay step. Isolation of adherence variants Ore adherence v a r i a n t s were obtained through n i t r o s o g u a n i d i n e (NG) mutagenesis (Adelberg, 1965) followed by s e v e r a l enrichment steps. The Ore ELISA (as described above) was used to screen the enriched c e l l p o p u l a t i o n f o r adherence-defective mutants. A s u r v i v a l curve was determined w i t h a constant c o n c e n t r a t i o n of NG and d i f f e r e n t time exposures. E x p o n e n t i a l l y growing c e l l s were harvested washed and resuspended i n c i t r a t e b u f f e r to an OD660 of 0.5. NG was added to a f i n a l c o n c e n t r a t i o n of 50 /^g/ml and t h i s was incubated at 37° C. At 20 min i n t e r v a l s c e l l s were removed, washed and resuspended i n phosphate b u f f e r and p l a t e d out on MSGYE media to determine the number of colony forming u n i t s (CFU). Mutagenized c e l l s were incubated f o r 24 hrs i n MSGYE l i q u i d media. The c e l l s were washed and resuspended i n adherence b u f f e r p r i o r t o enrichment. Each enrichment procedure c o n s i s t e d of two adsorption steps. The adsorption step i n v o l v e d a s e p t i c a l l y mixing 20 mis of the c e l l suspension (approx. l x l O 9 c e l l s per ml) with 1 gm of s t e r i l e Newmont mineral ore. The mixture was a g i t a t e d through continuous i n v e r s i o n i n a labquake f o r 20 min at 20° C. A f t e r i n c u b a t i o n the ore was removed by d i f f e r e n t i a l c e n t r i f u g a t i o n (2 min at 500xg) and the supernatant was recovered. A f t e r repeating once, the unbound c e l l s were resuspended i n MSGYE media and grown f o r three days at which time the next enrichment was performed. When enrichment was completed the c e l l s were p l a t e d and examined m a c r o s c o p i c a l l y and m i c r o s c o p i c a l l y . Selected c e l l s were screened f o r adherence a b i l i t y using the Ore ELISA assay, the Bio-Rad assay and a second i n d i r e c t method described by Takakuwa et a l . (197 9) i n which biomass i s measured by OD660 . C e l l envelope protein preparation C e l l s were washed i n adherence b u f f e r and resuspended i n a b u f f e r c o n t a i n i n g 0.05 M sodium phosphate, 0.15 M sodium c h l o r i d e and 0.01 M EDTA adjusted to pH 7.4 (Boyd & McBride, 1984). The c e l l suspension was sonicated (Branson Sonic Power Co., Danbury, Conn.) 3 times f o r 40 sec on pulsed mode followed by 10 sec on continuous mode. . The mixture was then c e n t r i f u g e d at 10,000*g f o r 20 min to remove unbroken c e l l s . The supernatant was recovered and c e n t r i f u g e d at 80,000xg f o r 2 hours. The p e l l e t of c e l l envelope p r o t e i n (CEP) was resuspended i n d i s t i l l e d water and i t s concentration was determined by the Bio-Rad p r o t e i n assay. C e l l envelope s o l u b i l i z a t i o n P r o t e i n s were s o l u b i l i z e d from the CEP pr e p a r a t i o n using v a r i o u s detergents; 0.1% Chaps, 0.1% T r i t o n X-100, 1 M l i t h i u m c h l o r i d e , 8 M urea, 5 M sodium thiocyanate, 6 M guanidine HCl, 0.1 M EDTA, 2.0% SDS or a combination of 8 M urea, 6 M guanidine HCl and 0.1 M EDTA. 50 j^q of CEP was added to 2 ml of each of the detergents. This mixture was incubated f o r 2 hours at 37° C and c e n t r i f u g e d f o r 2 hours at 80,000xg. The supernatant was d i a l y s e d overnight at 4° C against d i s t i l l e d water. A sample of CEP p r e p a r a t i o n was t r e a t e d w i t h d i s t i l l e d water and a f t e r c e n t r i f u g a t i o n , the supernatant and p e l l e t were separated to act as negative and p o s i t i v e c o n t r o l s r e s p e c t i v e l y . I n h i b i t i o n assay C e l l s were p r e t r e a t e d w i t h v a r i o u s agents and t e s t e d , by the Ore ELISA, t o determine t h e i r e f f e c t on c e l l adherence. These agents in c l u d e d ; p o l y s a c c h a r i d e s , p r o t e i n s , monovalent and d i v a l e n t c a t i o n s , reducing agents, b a c t e r i c i d a l agents, metal c h e l a t i n g agents and surface a c t i v e agents. C e l l s were washed and resuspended, to a concent r a t i o n of l x l O 9 c e l l s per ml, i n adherence b u f f e r c o n t a i n i n g one of the agents. The b a c t e r i a were incubated f o r 30 min at 20° C before dispensing 300 yul t o each Ore ELISA p l a t e w e l l . C e l l modification C e l l s were subjected t o enzymatic, p h y s i c a l and heat treatment to i n v e s t i g a t e t h e i r e f f e c t on adherence to min e r a l . The c e l l s were washed and resuspended, t o a concentration of l x l O 9 c e l l s per ml, 44 i n each treatment b u f f e r . Enzymatic treatment b u f f e r (50 mM T r i s , pH 7.2) contained e i t h e r ; 50 yi/g/ml phospholipase C, 50 yug/ml proteinase K, or 100 /^g/ml mixed g l y c o s i d a s e . Phospholipase C b u f f e r was supplemented w i t h 1 mM CaCl 2. For enzymatic treatment, c e l l s were incubated f o r 1 hr at 37° ,C. Heat treatment i n v o l v e d i n c u b a t i o n f o r ; 60 min at 50° C, 10 min at 70° C or 5 min at 100° C, i n 50 mM T r i s b u f f e r , pH 7.2. P h y s i c a l treatment i n v o l v e d v o r t e x i n g c e l l s w i t h a magnetic s t i r bar f o r 30 min i n 50 mM T r i s , pH 7.2 at 20° C. A f t e r treatment, c e l l s were washed and resuspended i n adherence b u f f e r f o r assay. Competition assay Competitive b i n d i n g assays were preformed w i t h immunologically d i s t i n c t c e l l types. The two c e l l types were mixed p r i o r to adding them to the Ore ELISA p l a t e . One c e l l type was maintained at a constant concentration while the other c e l l type was added i n i n c r e a s i n g c o ncentrations. The primary antibody used during the Ore ELISA assay was d i r e c t e d towards the c e l l s which were kept at constant c o n c e n t r a t i o n . Hydrophobicity assay B a c t e r i a l surface hydrophobicity was determined by measuring the number of b a c t e r i a adhering to hexadecane (Rosenberg et a l . , 1980; Ganeshkumar, 1985) . C e l l s were washed and resuspended i n adherence b u f f e r t o an absorbance at A 6 6 0 of 0.5. A volume of 0.1 ml hexadecane was added to 3.0 ml of c e l l suspension i n a 18x150 mm t e s t tube. The suspension was mixed on a vortex mixer f o r 60 sec at 10 sec i n t e r v a l s and allowed to stand f o r 20 min before reading. The c e l l s remaining i n the aqueous phase were c a r e f u l l y removed and A 6 6 0 values were determined. The t e s t samples were compared to c o n t r o l samples which were not t r e a t e d w i t h hexadecane. Surface protein preparation (SPP) C e l l s were washed and resuspended, t o a con c e n t r a t i o n of 5 x l 0 9 c e l l s per ml, i n a b u f f e r c o n t a i n i n g ; 20 mM T r i s , 150 mM NaCl, 10 mM MgCl 2, pH 7.2. This s o l u t i o n was mixed wi t h a magnetic s t i r bar f o r 30 min at 20° C. The c e l l s were removed by c e n t r i f u g a t i o n and the supernatant recovered. Forty percent ammonium sulphate was added to the supernatant and slowly s t i r r e d overnight at 4° C. The p r e c i p i t a t e was p e l l e t e d (80,000xg f o r 2 hrs) and resuspended i n d i s t i l l e d water. The pr e p a r a t i o n was ex h a u s t i v e l y d i a l y s e d against d i s t i l l e d water at 4° C and frozen at -20° C. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) SDS-PAGE, as described by Laemmli (1970), was performed w i t h a 10% or 12% polyacrylamide separating g e l and a 3% s t a c k i n g g e l . The samples were b o i l e d f o r 10 min i n SDS-^ME before adding approximately 0.3 y-<g of p r o t e i n per w e l l . The gels were s t a i n e d f o r p r o t e i n w i t h s i l v e r n i t r a t e as described by Oakley (1980). The molecular weight standards used were myosin (200,000), phosphorylase B (97,400), bovine ovalbumin (43, 000), OC -chymotrypsinogen (18,400) and lysozyme (14,300). serum albumin (68,000), (25, 700) , y 3 - l a c t o g l o b u l i n Preparation of IgG A n t i s e r a t o w i l d - t y p e A.cryptum and wild-type T.ferrooxidans c e l l s was prepared i n female New Zealand white r a b b i t s . C e l l s were washed i n d i s t i l l e d water and resuspended to a f i n a l c o n c e n t r a t i o n of l x l O 9 c e l l s per ml i n complete Freund's adjuvant. One ml of t h i s mixture was i n j e c t e d i n t r a m u s c u l a r l y on day 1. C e l l i n j e c t i o n s were repeated i n incomplete Freund's adjuvant on days 7 and 14. The r a b b i t s were b l e d on day 21 and immunoglobulin G (IgG) was obtained by p u r i f i c a t i o n on a P r o t e i n A-Sepharose CL4B (Sigma) column. Unbound serum c o n s t i t u e n t s were removed from the column w i t h borate b u f f e r (0.1 M borate, 0.5 M sodium c h l o r i d e , pH 8.4) u n t i l the e l u a t e was p r o t e i n f r e e . The bound IgG was e l u t e d w i t h g l y c i n e b u f f e r (0.1 M g l y c i n e , 0.5 M sodium c h l o r i d e , pH 2.5). The eluate was n e u t r a l i z e d w i t h d i l u t e NaOH and s t o r e d at -20° C. This antibody p r e p a r a t i o n w i l l be r e f e r r e d t o as the primary antibody through the r e s t of the t e x t . Adsorption of IgG Monospecific p o l y c l o n a l antiserum was prepared by adsorbing a n t i -whole c e l l a n t i s e r a t o adherence-defective v a r i a n t c e l l s . C e l l s were washed and resuspended i n PBS (pH 7.2) to a c o n c e n t r a t i o n of 5 x l 0 9 c e l l s per ml. 60 yal of IgG was added to 6 mis of c e l l 47 suspension and incubated overnight on a labquake at 4° C. The c e l l s were removed by c e n t r i f u g a t i o n and the supernatant was recovered. Adsorption was repeated four times. W e s t e r n b l o t Western b l o t a n a l y s i s was performed on samples th a t were electrophoresed i n 10% and 12% SDS-PAGE. The g e l was e q u i l i b r a t e d i n t r a n s f e r b u f f e r (14.4 gm/1 g l y c i n e , 3.0 gm/1 T r i s , 200 ml/1 methanol, pH 8.6) and placed on n i t r o c e l l u l o s e paper which had been wetted by immersion i n t r a n s f e r b u f f e r . The ge l and n i t r o c e l l u l o s e were sandwiched between two stacks of 3M Whatman f i l t e r paper which was a l s o wetted w i t h t r a n s f e r b u f f e r . This package was placed i n a h o l d i n g c a s s e t t e and immersed i n a Bio-Rad Trans-blot c e l l c o n t a i n i n g t r a n s f e r b u f f e r . To t r a n s f e r the p r o t e i n s t o the n i t r o c e l l u l o s e , a current of 25V was a p p l i e d overnight f o l l o w e d by a current of 60V f o r 2 hrs. The n i t r o c e l l u l o s e c o n t a i n i n g the t r a n s f e r r e d p r o t e i n s was immersed i n TBS (20 mM T r i s , 500 mM NaCl, pH 7.5) co n t a i n i n g 3% bovine serum albumin (BSA) f o r 30 min to block a l l remaining b i n d i n g s i t e s . The n i t r o c e l l u l o s e was incubated f o r 2 hrs i n primary antibody (see the l a s t two sections) d i l u t e d i n TBS-1% BSA. Unbound antibody was removed by washing twice i n TTBS (2 0 mM T r i s , 500 mM NaCl, 0.05% Tween-20, pH 7.5). The n i t r o c e l l u l o s e was then incubated f o r 2 hrs i n goat a n t i - r a b b i t IgG conjugated to horse r a d i s h peroxidase (GAR-HRP) d i l u t e d i n 48 TBS-1% BSA. Unbound conjugate antibody was removed by two washes i n TTBS. Bio-Rad GAR-HRP substrate was added and colour development was terminated by immersing the n i t r o c e l l u l o s e i n d i s t i l l e d water. Immunogold bead l a b e l l i n g C e l l s were washed and resuspended i n PBS to an A 6 6 0 of 0.5. Monospecific antibody (see "Adsorption of IgG" section) was added to the c e l l suspension and mixed i n a labquake at 4° C f o r 4 hrs. C e l l s were washed twice and resuspended i n 200 PBS. 15 j u l of a n t i - r a b b i t g o l d bead IgG (5 nm) was added and incubated overnight at 4° C i n a labquake. The c e l l s were then washed three times t o remove n o n - s p e c i f i c l a b e l l i n g . Electron microscopy C e l l s washed i n PBS were n e g a t i v e l y s t a i n e d w i t h 5% ur a n y l acetate i n 70% a l c o h o l . These were then observed under a P h i l i p s EM 300 e l e c t r o n microscope. SPP i n h i b i t i o n assay I n h i b i t i o n of c e l l adherence was performed using the SPP. Ten micrograms of SPP i n d i s t i l l e d water, from e i t h e r mutant-15 or the p a r e n t a l s t r a i n was added t o each w e l l and incubated f o r 30 min at 20° C. A f t e r r i n s i n g the p l a t e s w i t h adherence b u f f e r , 1x10 s c e l l s per ml of the parent s t r a i n were added t o each w e l l and the Ore ELISA was performed. The antibody c o n t r o l w e l l s were 49 t r e a t e d w i t h SPP but d i d not re c e i v e any c e l l s . Antibody i n h i b i t i o n assay Monospecific antibody was used to i n h i b i t A.cryptum adherence t o c h a l c o p y r i t e . P a r e n t a l s t r a i n c e l l s ( l x l O 9 c e l l s / m l ) were incubated overnight at 4° C i n PBS, pH 5.0, w i t h e i t h e r ; monospecific antibody or non-immune serum. C e l l s w i t h adsorbed antibody were washed w i t h and assayed i n adherence b u f f e r , pH 5.0. 50 RESULTS Bio-Rad assay f o r measuring A.cryptum adherence to chalcopyrite The Bio-Rad adherence assay was developed to c h a r a c t e r i z e A.cryptum adherence to c h a l c o p y r i t e ore. The design i s s i m i l a r to current i n d i r e c t methods of measuring m i c r o b i a l adherence which r e l y on p r o t e i n measurement as an i n d i c a t o r of biomass. However, i t was determined that p r o t e i n measurement using the Bio-Rad assay was f a s t e r and more s e n s i t i v e than the modified Lowry procedure (Lowry & Rosebrough, 1951). Some c h a r a c t e r i s t i c s of A.cryptum adherence t o c h a l c o p y r i t e are described i n Figures 1 through 4. Unless i n d i c a t e d otherwise, each Bio-Rad assay was performed using l x l O 8 A.cryptum c e l l s per ml and 50 mg of Newmont ore. The organisms were incubated w i t h the ore f o r 20 min. The adsorption isotherm ( F i g . 1) shows a l i n e a r r e l a t i o n s h i p between c e l l s added and c e l l s bound u n t i l s a t u r a t i o n was reached. S a t u r a t i o n occured when 1.2xl0 8 c e l l s were mixed w i t h 50 mg of ore. Figure 2 demonstrates the r e l a t i o n s h i p between the amount of Newmont ore and the number of c e l l s bound. 75 mg of ore bound the maximum number of c e l l s . At t h i s q u a n t i t y of ore, 51 14 2 -o H 1 1 1 1 1 1 1 1 — i 0 10 20 30 40 50 Cells Added (x10 7) Figure 1. Bio-Rad assay i l l u s t r a t i n g the adsorption of A.cryptum t o c h a l c o p y r i t e . C e l l were incubated f o r 20 min wi t h 50 mg of Newmont ore. 52 10 0 I • i — i — i — i i i i i i i — i i i — i — i — i — 0 20 40 60 80 100 120 Ore (mg) Figure 2. Bio-Rad assay demonstrating the r e l a t i o n s h i p between the number of c e l l s bound and the amount of Newmont ore added. A concentration of l x l 0 8 A.cryptum c e l l s were added and incubated f o r 20 min. 53 12 2H o H • 1—• 1 . 1 • — i • 1 1 2 3 4 5 6 pH Figure 3. Bio-Rad assay i l l u s t r a t i n g c e l l a d s orption as a f u n c t i o n of adherence b u f f e r pH. l x l O 8 c e l l s were added and incubated f o r 20 min with 50 mg of Newmont ore. 54 10 o -f • 1 • 1 1 • 0 10 20 30 40 Time (min) F i g u r e 4 . Bio-Rad assay i l l u s t r a t i n g the r e l a t i o n s h i p between i n c u b a t i o n time and the number of A.cryptum c e l l s bound. 1x10 s c e l l s were added t o 50 mg of Newmont ore. 55 approximately 15% of the 1 x 1 0 s c e l l s a v a i l a b l e f o r attachment, remained unbound. No increase i n b i n d i n g was observed when the qu a n t i t y of ore was increased t o 1 0 0 mg. The e f f e c t of pH on A.cryptum adherence t o c h a l c o p y r i t e was i n v e s t i g a t e d . The pH range t e s t e d corresponds wi t h that i n which A.cryptum c e l l s are v i a b l e . An inverse r e l a t i o n s h i p between adherence b u f f e r pH and a f f i n i t y to c h a l c o p y r i t e was recorded ( F i g . 3 ) . Figure 4 i l l u s t r a t e s the e f f e c t of in c u b a t i o n time on the number of c e l l s bound t o c h a l c o p y r i t e . Binding was r a p i d and complete i n 2 0 t o 30 min. " ' The age of the A. cryptum c u l t u r e had l i t t l e e f f e c t on c e l l adherence to c h a l c o p y r i t e . Maximum adherence was measured at 4 days. This l e v e l of adherence was maintained through to a 10 day c u l t u r e at which time c e l l s began to self-aggregate. There were a number of drawbacks encountered using the Bio-Rad assay f o r c e l l adherence: An i n i t i a l problem was the separation of ore f i n e s from suspended c e l l s . Adequate c e n t r i f u g a t i o n t o remove mineral f i n e s , a l s o removed unattached c e l l s from suspension. Conversely, l e s s s t r i n g e n t c e n t r i f u g a t i o n l e f t f i n e s and t h e i r attached b a c t e r i a i n suspension. The l o s s of f r e e l y suspended c e l l s was evaluated by the "spun" and "non-spun" 56 c o n t r o l s , and the removal of ore f i n e s i n suspension was measured by adsorbance. A compromise between these opposing f a c t o r s was reached w i t h a 20 sec s p i n at 5800xg. A second problem was the adsorption of b a c t e r i a l p r o t e i n to the gl a s s tubes during the assay which r e s u l t e d i n an over-estimation of bound c e l l s . F i n a l l y , the Bio-Rad assay was not a convenient means f o r screening l a r g e numbers of p o t e n t i a l adherence-defective mutants. These concerns provided a r a t i o n a l e f o r i n v e s t i g a t i n g an a l t e r n a t i v e approach f o r measuring adherence. Development o f the Ore ELISA The assay i s based on the same p r i n c i p l e as the standard ELISA except that a l a y e r of mineral ore i s glued to the w e l l surfaces of a f l a t - b o t t o m m i c r o t i t e r p l a t e . The ore acts as the receptor f o r b a c t e r i a l c e l l s ( F i g . 5 ). Ore attachment t o the ELISA p l a t e was s t a b l e provided the p l a t e was used w i t h i n three days f o l l o w i n g p r e p a r a t i o n . I t was found th a t a f t e r three days, ore p a r t i c l e s would become detached. To ensure that the w e l l s were thoroughly coated, each p l a t e was inspected under a d i s s e c t i n g microscope f o r voids i n the mineral surface. 5 7 Figure 5. C r o s s - s e c t i o n of an Ore ELISA p l a t e . 58 700 " 600 -500 -to o 400 -< • 300 -200 -100 -0 -Cells Added (x10 9) F i g u r e 6. E f f e c t of assay pH on adsorption at 4 05 nm. 59 The adherence b u f f e r , w i t h only t r a c e l e v e l s of s a l t s and a pH of 3.2, was designed to mimic the environment of t h i s a c i d o p h i l i c organism. An adherence b u f f e r pH of 3.2 was chosen because A.cryptum growth was highest i n media adjusted to t h i s pH. A number of parameters which could e f f e c t the assay were i n v e s t i g a t e d . Assay pH was an i n i t i a l concern, since A.cryptum i s an extreme a c i d o p h i l e and adherence to c h a l c o p y r i t e was shown to be i n v e r s e l y r e l a t e d to the pH (F i g . 3), i t was necessary that the assay be performed at an a c i d i c pH. However, i t was found that antibody r e a c t i v i t y was reduced at lower pHs. To address t h i s problem, the assay was performed at various assay pHs: The adherence b u f f e r f o r each assay was maintained at 3.2. However, the assay pH, which includes a l l other b u f f e r s of the assay (antibody b u f f e r , washing b u f f e r etc.) was v a r i e d . The r e s u l t s of t h i s experiment are shown i n Figure 6. An assay pH of 5.0 generated the highest OD405 values. Our i n t e r p r e t a t i o n of these r e s u l t s was tha t at pH 5.0 there was a balance between the desorption of c e l l s and the maintenance of enzyme and antibody r e a c t i v i t y . A 1/1000 d i l u t i o n of primary antibody stock s o l u t i o n was found to provide s u f f i c i e n t A 4 0 5 readings and y i e l d e d minimal background adsorption. A.cryptum d i d not possess an e x t r a c e l l u l a r a l k a l i n e phosphatase capable of r e a c t i n g w i t h the assay s u b s t r a t e . 60 A.cryptum showed strong a f f i n i t y f o r the p l a s t i c of the ELISA p l a t e and moderate a f f i n i t y f o r a glue coated ELISA p l a t e . Therefore i t was necessary to reduce background b i n d i n g to non-mineral surfaces. This was accomplished by extensive washing wi t h PBS/Tween. Adherence to ore coated p l a t e s , glue coated p l a t e s and untreated p l a s t i c p l a t e s was reduced by 5%, 65% and 82% r e s p e c t i v e l y by washing w i t h PBS/Tween. This suggests t h a t the c e l l s were i n t e r a c t i n g w i t h the ore as opposed t o the glue or the p l a t e . I n i t i a l t r i a l s of the Ore ELISA produced s u b s t a n t i a l background readings due t o n o n - s p e c i f i c adsorption of the antibody to the mineral ore sub s t r a t e . Table I I i l l u s t r a t e s the procedures employed t o reduce the background l e v e l s ; these p r i m a r i l y i n v o l v e d a l t e r i n g the washing or b l o c k i n g steps. A s i x percent reduction i n background was achieved by i n t r o d u c i n g a b l o c k i n g step p r i o r t o adding the primary antibody. Increasing the concent r a t i o n of BSA or using other p r o t e i n b l o c k i n g agents (data not included) during the b l o c k i n g step had l i t t l e e f f e c t on f u r t h e r i n g background r e d u c t i o n . The most e f f e c t i v e assay c o n d i t i o n f o r maintaining low background l e v e l s was a 1 hr block w i t h 1% BSA and a f o r 10 min wash w i t h PBS/Tween. This procedure reduced background l e v e l s from 32% t o 12%. To r e l a t e A 4 0 5 readings to the number of c e l l s , the r e a c t i v i t y of the c e l l s w i t h antibody was assessed by performing the tube assay concurrently with each experiment. This procedure served to standardize A 4 0 5 assay readings such that adherence comparisons between d i f f e r e n t c e l l types, species and s tra ins could be made. For an assay to be use fu l , i t must be s p e c i f i c , reproducable and s e n s i t i v e . Table III indicates that the assay i s dependent upon having a s p e c i f i c antibody and that non-spec i f i c adsorption of antibody to ore i s minimal. R e l i a b i l i t y was assessed by measuring the v a r i a b i l i t y of readings between and within assay p l a t e s . Each experiment performed on the Ore ELISA was run i n t r i p l i c a t e . Readings from d i f f erent wells on the same p late var i ed between 3% and 7%. Assay s e n s i t i v i t y was assessed by measuring A 4 0 5 at various c e l l concentrat ions. A 100% increase over background was observed with a minimum of 1x10s c e l l s . The Ore ELISA was appl ied to adherence k i n e t i c studies and the resu l t s were compared to the Bio-Rad assay r e s u l t s . The adsorption isotherms for the two assays both displayed binding saturat ion (F ig . 1 & 7) . Binding saturat ion for the Ore ELISA was found to be 1.4xl0 8 c e l l s which i s s i m i l a r to the 1.2xl0 8 c e l l s recorded for the Bio-Rad assay. The ef fect of pH on adherence as measured by the Ore ELISA was less marked than that shown by the Bio-Rad assay (Fig . 3 & 8) . 62 Table I I . E f f e c t of Tween and BSA on background-reduction i n the Ore ELISA assay. A s s a y conditions A 4 0 5 B S A block Tween wash Ab contro l 1 Test^ Background^ - - .580 1.807 32 - + .456 1.683 27 1% + .335 1.562 21 2 % + .331 1.400 23 3 % + .293 1.367 22 1% + 4 .155 1.294 12 1. A s s a y performed without cel ls added . 2. A s s a y performed with 1x1 09 ce l ls added. 3. Background was calculated as the percent of antibody control relat ive to the test. 4. The final P B S / T w e e n wash was for 10 min. 63 Table I I I . Immunological s p e c i f i c i t y of the Ore ELISA assay. A s s a y Condit ion &405 Without ce l l s a d d e d 0 . 1 4 4 Without a n t i - A . c r v p t u m A b 0 . 0 7 8 Without seconda ry Ab 0 . 0 2 7 Without a lka l ine phosphatase substrate 0.005 Comp le te a s s a y with an t i -A .c ryp tum A b 1.350* Comple te a s s a y with a n t i - T . f e r r o o x i d a n s Ab 0.101 * A405 of 1.350 represents 1.0x1 08 ce l ls bound. 64 C e l l s bound r a p i d l y , adherence was e s s e n t i a l l y complete i n 10 min (Fi g . 9). The f l a t p l ateau of Figure 10 would suggest t h a t c e l l s adhere i n a monolayer and do not aggregate. This was supported by microscopic s t u d i e s i n which c e l l aggregation was not observed. Adherence t o d i f f e r e n t mineral ore substrates was i n v e s t i g a t e d . The greatest number of A.cryptum c e l l s bound to the Cambell Red lake ore sample w i t h twenty f i v e percent fewer b i n d i n g to the Empire mines ore sample ( F i g . 10). Attempts at using elemental sulphur as the receptor proved unsuccessful as the sulphur d i d not form a s t a b l e homogenous l a y e r i n the m i c r o t i t e r p l a t e . Adherence i n h i b i t i o n studies The i n h i b i t o r y e f f e c t of va r i o u s agents was i n v e s t i g a t e d , i n an attempt to i d e n t i f y the mechanism of adherence. These agents were present i n the adherence b u f f e r during the b i n d i n g r e a c t i o n . The adherence recorded i n Tables IV through IX was c a l c u l a t e d as a percent, assuming that the number of c e l l s bound i n the c o n t r o l represents 100%. N-acetyl glucosamine, N-acetyl galactosamine and a number of other sugars t e s t e d had no e f f e c t on A.cryptum 65 F i g u r e 7. Ore ELISA measuring the adsorption isotherm f o r A.cryptum b i n d i n g to c h a l c o p y r i t e . 66 2 -o H—i—i—•—i—i—i—•—i—<—i—•—i—«— 0 1 2 3 4 5 6 7 pH F i g u r e 8 . Ore ELISA demonstrating the e f f e c t of adherence b u f f e r pH on A.cryptum b i n d i n g t o c h a l c o p y r i t e . 67 12 Time (min) Figure 9. R e l a t i o n s h i p between length of in c u b a t i o n and adherence as measured i n the Ore ELISA assay. 68 F i g u r e 10. A comparison of A.cryptum adherence t d i f f e r e n t m i n e r a l s . adherence to c h a l c o p y r i t e (Table IV) . On the other hand, both BSA and g e l a t i n i n h i b i t e d c e l l b i n d i n g to the ore (Table V). For example, 0.1 mg/ml of BSA or g e l a t i n reduced c e l l adherence by 25% and 67% r e s p e c t i v e l y . Though t h i s form of i n h i b i t i o n appeared n o n - s p e c i f i c i n nature, the e f f e c t of g e l a t i n was greater than t h a t of BSA. That i s , a 10 f o l d increase i n BSA was necessary to achieve an equivalent l e v e l of i n h i b i t i o n . In the presence of monovalent and d i v a l e n t c a t i o n s , A.cryptum adherence t o c h a l c o p y r i t e was enhanced (Table V I ) . However, t h i s phenomenon was only observed at high i o n concentrations. Surface a c t i v e agents g r e a t l y enhanced the adherence of A.cryptum to c h a l c o p y r i t e (Table V I I ) . For example, T r i t o n X-100 at a 0.01% concentr a t i o n increased c e l l adherence by 310%. The e f f e c t of c h e l a t i n g agents, b a c t e r i c i d a l agents and reducing agents are summarized i n Table V I I I . The c e l l suspension was incubated w i t h the chemical agent f o r 30 min at 2 0° C and then added t o the Ore ELISA p l a t e . E l e v a t e d adherence was recorded i n the presence of the c h e l a t i n g agent, EDTA. 2-mercaptoethanol and DTT increased and decreased adherence r e s p e c t i v e l y . The sulphur b i n d i n g agents, iodoacetamide and mercuric c h l o r i d e , had l i t t l e e f f e c t on adherence. The r e s p i r a t o r y poisons, sodium azide and sodium cyanide had a minimal e f f e c t on adherence suggesting that c e l l v i a b i l i t y was not necessary f o r adherence (Table V I I I ) . 70 Table IV. The e f f e c t of polysaccharides on A.cryptum adherence to chalcopyrite. Cone. (mM) Adherence1 Control 2 100 Glucose 10 113 100 110 Galactose 10 88 100 99 Lactose 10 93 100 100 Mannose 10 111 100 104 Ribose 10 101 100 73 N-Acetyl Glc 10 108 100 116 N-Acetyl Gal 10 99 100 109 1. Percent of cells bound relative to the control. 2. Cells incubated in adherence buffer. 71 Table V. The e f f e c t of p r o t e i n on A.cryptum adherence t o c h a l c o p y r i t e . Cone, ( m g / m l ) Adherence 1 C o n t r o l 2 1 0 0 B S A 0.01 93 0.1 75 1 37 G e l a t i n 0.01 73 0.1 33 1 11 1. Percent of ce l ls bound relative to the control . 2 . Ce l l s incubated in adherence buffer. 72 Table V I . The e f f e c t of monovalent and d i v a l e n t c a t i o n s on A.cryptum adherence t o c h a l c o p y r i t e . Cone. (mM) Adherence 1 C o n t r o l 2 100 N a C l 10 114 100 111 500 156 M g S 0 4 5 105 20 112 100 120 C a C I 2 5 109 20 114 100 129 1. Percent of ce l ls bound relative to the c o n t r o l 2. Ce l l s incubated in adherence buffer. 73 Table VII. The e f f e c t o f s u r f a c e a c t i v e a g e n t s on A.cryptum adherence t o c h a l c o p y r i t e . Agent Percent Adherence C o n t r o l 2 1 0 0 Tween 20 0.001 1 2 3 0.01 2 1 6 0.1 2 1 4 Tri ton X - 1 0 0 0.001 1 2 3 0.01 3 1 0 0.1 2 9 0 1. Percent of ce l ls bound relative to the c o n t r o l . 2. Ce l ls incubated in adherence buffer. 74 Table VIII. The e f f e c t of reducing agents, b a c t e r i c i d a l agents and metal c h e l a t i n g agents on A.cryptum adherence to c h a l c o p y r i t e . Agent Cone. (mM) Adherence 1 C o n t r o l 2 1 0 0 Iodoace tamide 1.0 1 1 6 Mercur ic Ch lo r ide 1.0 1 0 4 2 - M e r c a p t o e t h a n o l 1.0 1 4 8 D i t h i o t h r e i t o l 1.0 74 Sod ium az ide 2.0 93 S o d i u m fe r r i cyana te 0.1 23 Sod ium cyan ide 2.0 121 EDTA 2.0 1 5 3 1. Percent of ce l l s bound relative to the control. 2. Ce l l s incubated in adherence buffer. 75 Sodium f e r r i c y a n a t e , u n l i k e t h e o t h e r b a c t e r i c i d a l a g e n t s , s t r o n g l y r e d u c e d c e l l adherence. I t s h o u l d be n o t e d t h a t i n t h e pr e s e n c e o f sodium f e r r i c y a n a t e , t h e s o l u t i o n t u r n e d a v i v i d b l u e c o l o u r . T h i s was p r o b a b l y due t o t h e c h e m i c a l l e a c h i n g o f copper from c h a l c o p y r i t e by f e r r i c y a n a t e . In an attempt t o de t e r m i n e i f i n h i b i t o r s o f b i n d i n g were a c t i n g on t h e c e l l s o r t h e o r e , e x p e r i m e n t s were run i n whi c h t h e i n h i b i t o r was p r e i n c u b a t e d f o r 30 min w i t h e i t h e r t h e o r e o r t h e c e l l s and t h e n removed p r i o r t o t h e assay (Table IX, column 1 & 2) . The r e s u l t s were compared t o as s a y s i n whi c h t h e i n h i b i t o r was p r e s e n t d u r i n g t h e assay (Table IX, column 3 ) . EDTA p r e t r e a t m e n t o f e i t h e r t h e ore or t h e c e l l s had no e f f e c t on adherence but i f p r e s e n t d u r i n g t h e adherence r e a c t i o n , b i n d i n g was i n c r e a s e d . Sodium f e r r i c y a n a t e seems t o a c t p r i m a r i l y a t t h e c e l l s u r f a c e , however, an a c c u m u l a t i v e e f f e c t may be n e c e s s a r y t o reduce adherence t o 46% (Table IX, column 3) . D i t h i o t h r e i t o l p r e t r e a t m e n t had l i t t l e e f f e c t on c e l l attachment t o c h a l c o p y r i t e . However, i f DTT was p r e s e n t d u r i n g t h e adherence r e a c t i o n , b i n d i n g was d e c r e a s e d . S i m i l a r t o t h e e f f e c t s o f EDTA, th e p r e s e n c e o f 2-mercaptoethanol i n t h e adherence b u f f e r was n e c e s s a r y f o r t h e ob s e r v e d i n c r e a s e i n b i n d i n g . The e f f e c t o f c e l l s u r f a c e m o d i f i c a t i o n on adherence was i n v e s t i g a t e d . P h o s p h o l i p a s e C, p r o t e i n a s e K and mixed g l y c o s i d a s e had no e f f e c t on A.cryptum adherence t o c h a l c o p y r i t e . Table IX. The e f f e c t of ore or c e l l pretreatment w i t h reducing agents, b a c t e r i c i d a l agents and metal c h e l a t i n g agents on A.cryptum adherence to c h a l c o p y r i t e . Pretreatment of ore Pretreatment of cel ls Cel ls in treatment buffer C o n t r o l 1 1 0 0 100 100 2 - M e r c a p t o e t h a n o l 98 79 152 D i t h i o t h r e i t o l 90 90 64 S o d i u m fer r icyanate 83 72 46 Sod ium cyan ide 79 88 104 EDTA 96 96 124 Al l va lues calcu lated as a percent of ce l ls bound relative to the c o n t r o l . 1. Ce l l s incubated in adherence buffer. 77 The e f f e c t of heat could not be assessed as the c e l l s formed aggregates when heated. C e l l s which had been mixed vigorously with a s t i r bar retained the a b i l i t y to bind to chalcopyrite. Competition studies A.cryptum and T.ferrooxidans are c l o s e l y linked i n nature and are believed to exis t symbiotically. Of interest was whether these two acidophiles compete for binding s i t e s on chalcopyrite. This type of experiment was possible using the Ore E L I S A assay because the two species c e l l types are immunologically d i s t i n c t and do not coaggregate as shown by microscopic studies. Table I I I , i n addition to immunofluorescence studies, demonstrates that antisera to A.cryptum and T.ferrooxidans are not cross-reactive. The competition experiment was designed to maintain one species at a constant saturating concentration and increase the concentration of the second species. This protocol would provide comparative information about the nature and s p e c i f i c i t y of adherence of the two c e l l types. Saturation was achieved with 3xl0 9 A.cryptum cell/ml (Fig. 7) and 2xl0 9 T.ferrooxidans cell/ml (Fig. 11). Figure 12 (a) demonstrates that a 20 f o l d excess of A.cryptum c e l l s d i d not in t e r f e r e with T.ferrooxidans binding to chalcopyrite. A sim i l a r result was recorded for the binding of 78 30 o H — i — i — i — i — • — i — • — i — • — i — • — I 0 10 20 30 40 50 60 Cells Added (x10 8) Figure 11. Ore E L I S A m e a s u r i n g t h e b i n d i n g o f T.ferrooxidans t o c h a l c o p y r i t e . 79 a) o X T3 c o J3 o u CO c eg T3 x o o 0 5 10 15 20 A.crvptum/T.ferrooxldans 25 b) o X T3 c 3 o .fl <t> o E >. v> O < 0 5 10 15 20 25 T.ferrooxIdans/A.cryptum F i g u r e 12. Competition between A.cryptum and T. ferrooxidans f o r b i n d i n g s i t e s on c h a l c o p y r i t e : a) Increasing numbers of A.cryptum c e l l s were added t o a constant number of T.ferrooxidans c e l l s (2xl0 9 c e l l s / m l ) . b) Increasing numbers of T. ferrooxidans c e l l s were added to a constant number of A.cryptum c e l l s (3xl0 9 c e l l s / m l ) . 80 A.cryptum t o c h a l c o p y r i t e i n the presence of an excess of T. ferrooxidans ( F i g . 12 (b)) . I t was concluded t h a t the b i n d i n g s i t e s f o r each organism are unique and d i s t i n c t . Isolation of adherence-defective variants In order to i d e n t i f y the adhesin r e s p o n s i b l e f o r attachment to mineral surfaces, A.cryptum c e l l s were mutagenized, enriched and screened f o r adherence-defective mutants. Each of three mutagenesis experiments performed used a s t a r t i n g c o n c e n t r a t i o n of 3 x l 0 8 c e l l s per ml and 50 / A g / m l of n i t r o s o g u a n i d i n e . The f i r s t mutagenesis was aimed at a 50% k i l l according to the s u r v i v a l curve ( F i g . 13). This r e q u i r e d a 47 min exposure to the mutagen and was followed by a s e r i e s of 6 enrichment procedures. In a second and t h i r d experiment c e l l s were exposed to NG f o r 80 min and enriched f o r non-adherent c e l l s . C e l l s were enriched 6 and 14 times f o l l o w i n g second and t h i r d enrichments r e s p e c t i v e l y . In these experiments, enrichment i n v o l v e d mixing mutagenized c e l l s w i t h ore f o r 20 min and then c u l t u r i n g c e l l s which d i d not s e t t l e w i t h the ore. The f i r s t two attempts at o b t a i n i n g non-adherent mutants were unsuccessful. Assuming t h a t b i n d i n g may i n v o l v e more than one type of receptor-adhesin i n t e r a c t i o n , i t was decided to modify the t h i r d mutagenesis enrichment procedure i n an attempt t o d i f f e r e n t i a t e between weak and strong b i n d i n g 81 0 H r—i J l i 1 1 r—] r — i 1 1 • 1 • • 1 0 20 40 60 80 100 120 Time (min) Figure 13. N i t r o s o g u a n i d i n e s u r v i v a l curve. 82 c e l l s . Instead of a 20 min i n c u b a t i o n w i t h 1 gm of Newmont ore, the c e l l suspension was incubated w i t h e i t h e r 25 mg of ore f o r 20 min, or 100 mg of ore f o r 10 sec. The modified enrichments would s e l e c t f o r b i n d i n g strength and b i n d i n g speed r e s p e c t i v e l y . These enrichments were performed 6 times, the c e l l s remaining i n suspension were p l a t e d and c o l o n i e s screened f o r t h e i r a b i l i t y to b i n d t o c h a l c o p y r i t e . I s o l a t e s were screened by the Ore ELISA and recorded as the percent of adherent c e l l s r e l a t i v e t o the number of parent c e l l s bound. I n d i r e c t assays confirmed the Ore ELISA screening r e s u l t s . P r i o r t o screening, the c e l l c o n c e n t r a t i o n of the mutant and parent s t r a i n s was adjusted to l x l O 9 c e l l s per ml. Each mutant was t e s t e d f o r antibody r e a c t i v i t y by the tube assay to ensure that mutagenesis d i d not create q u a n t i t a t i v e changes i n the r e a c t i v i t y of the c e l l s to the antibody. Several of the mutants proved to be s e l f - a g g r e g a t i n g and were not f u r t h e r s t u d i e d . Table X l i s t s s e v e r a l of the mutants i s o l a t e d . A s t r i c t l y non-adherent mutant was not obtained. Most mutants d i s p l a y e d a 41-61% decrease i n a b i l i t y t o adhere to c h a l c o p y r i t e . However, mutant-4 showed an increase i n the a b i l i t y to b i n d t o c h a l c o p y r i t e . Mutants -30, -39, -76, and -90 were i s o l a t e d from 83 Table X. C h a r a c t e r i s t i c s of adherence-defective mutants. Strain Adhe rence 1 Hydrophobic i ty 2 Self-aggregation Parental 100 86 -4 128 85 _ 11 40 83 -12 51 78 -15 51 89 -19 43 65 + 23 40 69 + 30 52 77 -39 58 48 + 76 59 58 + 90 39 40 + 1. Percen t of ce l ls bound relative to the control . 2. Percent of cel ls remaining in the aqueous phase. 84 the f i r s t mutagenesis, the t h i r d mutagenesis, any of the i s o l a t e s . The remaining mutants were i s o l a t e d from Phenotype r e v e r s i o n was not detected i n Analysis of adherence-defective mutants Many studie s have c o r r e l a t e d c e l l surface hydrophobicity w i t h adherence t o mineral surfaces and to i n t e r f a c e s i n the environment. However, i n these s t u d i e s , a r e l a t i o n s h i p between c e l l surface hydrophobicity and the a b i l i t y to b i n d to ore was not observed. For example, mutant -90 i s hydrophobic and mutant-15 i s h y d r o p h i l i c yet both are d e f e c t i v e i n t h e i r a b i l i t y to adhere t o c h a l c o p y r i t e . Hydrophobic c e l l s appeared t o s e l f -aggregate . Many microorganisms a t t a c h to surfaces v i a p o l a r or l a t e r a l f l a g e l l a . This d i d not appear to be the case w i t h A. cryptum since the adherence-defective mutants possessed f l a g e l l a and are m o t i l e . Western immunoblot a n a l y s i s of whole c e l l l y s a t e s showed th a t a 31.6 kDa antigen present i n the parent s t r a i n ( F i g . 14, Lane A), was absent from the adherence-defective mutants ( F i g . 14, Lanes D, E, & F ) . Mutant-4 ( F i g . 14, Lane C) which showed enhanced 8 5 Figure 14. Western b l o t o f a 10% SDS-PAGE d e v e l o p e d w i t h anti - A . c r y p t u m a n t i b o d y . Whole c e l l l y s a t e s o f : Lane A and B, p a r e n t a l A.cryptum. Lane C, mutant-4 which showed enhanced adherence a b i l i t y . Lanes D, E, and F are a d h e r e n c e - d e f e c t i v e mutants-11,-12, and -15 r e s p e c t i v e l y . Samples i n l a n e s A, C-F, were h e a t e d t o 100° C i n SDS p r i o r t o e l e c t r o p h o r e s i s . The sample i n l a n e B was i n c u b a t e d i n SDS a t 20° C p r i o r t o e l e c t r o p h o r e s i s . 86 adherence a b i l i t y , a l s o contained the 31.6 kDa antigen. B o i l i n g the sample had no e f f e c t on the migra t i o n p a t t e r n of the 31.6 kDa antigen ( F i g . 14, Lane A & B) . An a d d i t i o n a l low molecular weight antigen, common to a l l mutants, was i d e n t i f i e d i n Figure 14. The antigen m i g r a t i o n p r o f i l e of each mutant c l o s e l y resembles the p a r e n t a l s t r a i n , suggesting that the mutants are not contaminants. This was supported by microscopic and macroscopic comparisons. The 31.6 kDa p r o t e i n was absent i n a l l the adherence-defective mutants t e s t e d . The p r o t e i n was present i n p a r e n t a l c e l l s harvested from l i q u i d and s o l i d media. C e l l s from e i t h e r medium had equivalent ore adherence a b i l i t i e s . L o c a l i z a t i o n of the 31.6 kDa protein Since the 31.6 kDa p r o t e i n was absent i n adherence-defective s t r a i n s , and present i n p a r e n t a l and adherence-enhanced s t r a i n s , i t was p o s t u l a t e d that the p r o t e i n might be i n v o l v e d i n the bin d i n g of A.cryptum to c h a l c o p y r i t e . Therefore, attempts were made t o l o c a l i z e the 31.6 kDa p r o t e i n . On the assumption that l o o s e l y a s s o c i a t e d c e l l surface s t r u c t u r e s could be removed by gentle means, we vortexed c e l l s w i t h a magnetic s t i r bar and analysed the m a t e r i a l s o l u b i l i z e d by t h i s process (surface p r o t e i n p r e p a r a t i o n ) . 87 F i g u r e 15. S D S - P A G E o f s u r f a c e p r o t e i n p r e p a r a t i o n s . Lane A , whole c e l l l y s a t e o f p a r e n t a l A.cryptum. Lane B , c e l l e n v e lope p r o t e i n p r e p a r a t i o n o f p a r e n t a l A.cryptum. Lane C, D , and E , s u r f a c e p r o t e i n p r e p a r a t i o n s o f p a r e n t a l A.cryptum, mutant-12, and mutant-15 r e s p e c t i v e l y . Lane A c o n t a i n s 0.3 g o f p r o t e i n . Lane B c o n t a i n s 0.2 / j g o f p r o t e i n . Lanes C, D , and E each c o n t a i n 0.05/^g o f p r o t e i n . 88 F i g u r e 16. Western b l o t o f a 12% SDS-PAGE d e v e l o p e d w i t h anti-A.cryptum a n t i b o d y . Lane A, whole c e l l l y s a t e o f p a r e n t a l A.cryptum. Lane B, c e l l e nvelope p r o t e i n p r e p a r a t i o n o f p a r e n t a l A.cryptum. Lane C, D, and E, s u r f a c e p r o t e i n p r e p a r a t i o n s o f p a r e n t a l A.cryptum, mutant-12, and mutant-15 r e s p e c t i v e l y . 89 Figure 15 i s a s i l v e r s t a i n e d SDS-PAGE comparing the m i g r a t i o n p r o f i l e of; p a r e n t a l whole c e l l l y s a t e (Lane A) , p a r e n t a l c e l l envelope p r o t e i n p r e p a r a t i o n (Lane B) and p a r e n t a l surface p r o t e i n p r e p a r a t i o n (Lane C) . The absence of the 31.6 kDa p r o t e i n i s i l l u s t r a t e d i n the surface p r o t e i n preparations of mutants-12 and -15, lanes D and E r e s p e c t i v e l y . Figure 16 i s a western immunoblot of Figure 15 and f u r t h e r suggests t h a t the 31.6 kDa p r o t e i n i s l o c a l i z e d at the c e l l surface of the p a r e n t a l s t r a i n and i s absent i n the mutant s t r a i n s . The v a r y i n g i n t e n s i t y of the 31.6 kDa p r o t e i n between preparations i n Figure 15 was d i s t u r b i n g . Though f i g u r e 16 shows s i m i l a r i n t e n s i t i e s of banding suggesting s i m i l a r antigen concentrations, a d i s t i n c t r e duction i n banding i n t e n s i t y i s observed i n f i g u r e 15. There are two p o s s i b l e explanations f o r t h i s discrepancy. The t i t e r of antibody s p e c i f i c to the 31.6 kDa p r o t e i n may be l i m i t e d i n the anti-A.cryptum a n t i s e r a and t h e r e f o r e would not r e f l e c t antigen c o n c e n t r a t i o n d i f f e r e n c e s during western immunoblot a n a l y s i s . An a l t e r n a t i v e e x p l a n a t i o n i s the presence of a s i m i l a r s i z e d p r o t e i n i n the whole c e l l l y s a t e w i t h was removed during the c e l l envelope p r o t e i n p r e p a r a t i o n . A monospecific p o l y c l o n a l antibody p r e p a r a t i o n was developed i n order to l o c a l i z e the 31.6 kDa p r o t e i n through immunogold l a b e l l i n g . A n t i s e r a t o the p a r e n t a l s t r a i n of A.cryptum was thoroughly adsorbed by the mutant-15 to generate the monospecific antibody p r e p a r a t i o n i l l u s t r a t e d i n Figure 17. Several 90 conclusions can be made from t h i s f i g u r e : The adsorption procedures generated antibody w i t h a s i n g l e s p e c i f i c i t y . Further evidence was provided t o suggest that s i m i l a r concentrations of the 31 .6 kDa antigen e x i s t i n whole c e l l l y s a t e s and c e l l envelope preparations of the p a r e n t a l s t r a i n ( F i g . 17, Lanes A & B) . The m o n o - s p e c i f i c i t y of the adsorbed antibody suggests that mutant-15 and the p a r e n t a l s t r a i n s d i f f e r only i n the 31 .6 kDa p r o t e i n . Mutant-15 and the p a r e n t a l s t r a i n were immunogold l a b e l l e d using the antibody p r e p a r a t i o n s p e c i f i c t o the 31 .6 kDa p r o t e i n . Figure 18 shows an even d i s t r i b u t i o n of gold beads i n d i c a t i n g t h a t the antigen was widely d i s t r i b u t e d over the surface of the p a r e n t a l s t r a i n . This was compared t o mutant-15 l a b e l l e d w i t h the monospecific antibody i n Figure 19 (a) , and the p a r e n t a l s t r a i n l a b e l l e d w i t h non-immune serum i n Figure 19 (b) . The i n t e n s i t y of gold bead l a b e l l i n g i n Figure 18 was not c o n s i s t e n t w i t h a l l p a r e n t a l c e l l s observed; some contained very few gold beads while others showed excessive l a b e l l i n g . This may be i n d i c a t i v e of phase v a r i a t i o n i n the p r o t e i n expression of the 31 .6 kDa p r o t e i n . 91 A B C D 9 7 . 4 -68-43--31.6 25.7-F i g u r e 17. Western b l o t o f a 12% SDS-PAGE d e v e l o p e d w i t h t h e m o n o s p e c i f i c a n t i - 3 1 . 6 kDa p r o t e i n p o l y c l o n a l a n t i b o d y p r e p a r a t i o n . Lane A, c e l l e n v e lope p r o t e i n p r e p a r a t i o n o f p a r e n t a l A.cryptum. Lane B, whole c e l l l y s a t e o f p a r e n t a l A. cryptum. Lane C , and D, c e l l e n v e l ope p r o t e i n p r e p a r a t i o n and whole c e l l l y s a t e o f mutant-15 r e s p e c t i v e l y . 9 2 V a r i a t i o n s i n the stringency of washing during immunogold l a b e l l i n g generated marked d i f f e r e n c e s i n the numbers of gol d beads l o c a t e d on the c e l l surface. Gentle c e l l resuspension using a pasteur p i p e t t e r e s u l t e d i n c o n s i s t e n t l a b e l l i n g as shown i n Figure 18. However, more vigorous washing i n a vortex mixer generated a l e s s evenly d i s t r i b u t e d p a t t e r n of gold beads as i l l u s t r a t e d i n Figure 20. In t h i s case, the 31.6 kDa p r o t e i n appeared to form aggregates on and away from the p a r e n t a l c e l l surfaces as i n d i c a t e d by the clumps of beads ( F i g . 20, arrow). The apparent loose a s s o c i a t i o n of the 31.6 kDa p r o t e i n w i t h the c e l l surface was a l s o shown i n Figure 21. In t h i s case the g o l d beads appeared to be deposited i n the shape of an A.cryptum c e l l suggesting that the 31.6 kDa p r o t e i n was adsorbed to the e l e c t r o n microscope g r i d and l e f t behind a f t e r the c e l l s were sheared away. Having e s t a b l i s h e d t h a t the 31.6 kDa p r o t e i n was a s s o c i a t e d w i t h the c e l l envelope, s o l u b i l i z a t i o n s t u d i e s were performed i n an attempt to p u r i f y the p r o t e i n . C e l l envelope p r o t e i n preparations were t r e a t e d w i t h detergents and d i f f e r e n t i a l l y c e n t r i f u g e d to separate the s o l u b i l i z e d p r o t e i n from the remaining envelope-bound p r o t e i n s . The s o l u b i l i z e d p r o t e i n s were subjected to western immunoblot a n a l y s i s . Only very f a i n t 93 F i g u r e 18. Immunogold bead l a b e l l i n g o f p a r e n t a l A.cryptum c e l l s w i t h a n t i - 3 1 . 6 kDa a n t i b o d y f o l l o w e d by u r a n y l a c e t a t e n e g a t i v e s t a i n i n g . Bar = 0.5 u i . 94 F i g u r e 19 (a) . Immune-gold bead l a b e l l i n g of mutant-15 wit h anti-31.6 kDa p o l y c l o n a l antibody followed by urany l acetate negative s t a i n i n g . Bar = 0.5 ^m. 95 Figure 19 (b) . Immune-gold bead l a b e l l i n g o f p a r e n t a l A.cryptum w i t h non-immune serum f o l l o w e d by u r a n y l a c e t a t e n e g a t i v e s t a i n i n g . Bar = 1 yum. 96 F i g u r e 20. Immunogold bead l a b e l l i n g of parental A.cryptum c e l l s with anti-31.6 kDa polyclonal antibody followed by uranyl acetate negative staining. C e l l s were mixed vigorously during the washing steps. The arrow indicates 31.6 kDa protein aggregates at the c e l l surface. Bar = 1 yum. 97 F i g u r e 21. Immune-gold bead l a b e l l i n g of a p a r e n t a l s t r a i n A.cryptum c e l l p r i n t w i t h anti-31.6 kDa p o l y c l o n a l antibody. Bar = 1 yum. 98 banding was observed through treatment w i t h guanidine HCl, urea or combined guanidine HCl-urea-EDTA and as such, was not considered an e f f e c t i v e approach to p u r i f i c a t i o n . I n h i b i t i o n assays were attempted using the monospecific antibody or the 31.6 kDa surface p r o t e i n p r e p a r a t i o n but the r e s u l t s were i n c o n c l u s i v e . A f t e r t r e a t i n g the c e l l s w i t h the monospecificantibody, the c e l l s formed l a r g e aggregates. T r e a t i n g the p l a t e s w i t h the surface p r o t e i n preparations r e s u l t e d i n high background readings and as such l i m i t e d the usefulness of t h i s technique. 99 DISCUSSION The purpose o f t h i s r e s e a r c h was t o e s t a b l i s h t e c h n i q u e s f o r s t u d y i n g a c i d o p h i l e adherence to m i n e r a l s u r f a c e s . T h i s i s the f i r s t s tudy on the adherence p r o p e r t i e s o f the a c i d o p h i l i c h e t e r o t r o p h , A.cryptum. The r e s u l t s c l e a r l y demonstrate t h a t t h i s organism b i n d s t o c h a l c o p y r i t e and p y r i t e . B i n d i n g was demonstrated by assays which measured c e l l p r o t e i n and r e a c t i v i t y w i t h a s p e c i f i c a n t i b o d y . The a c i d o p h i l i c h e t e r o t r o p h s are thought t o be impor tant e c o l o g i c a l p a r t n e r s i n T.ferrooxidans c o l o n i z a t i o n o f m i n e r a l s u r f a c e s . The h e t e r o t r o p h s consume o r g a n i c a c i d s and o t h e r m i c r o b i a l m e t a b o l i t e s which are t o x i c to the n u t r i t i o n a l l y f a s t i d i o u s a u t o t r o p h s . T h i s type o f in terdependence would b e n e f i t from a c l o s e a s s o c i a t i o n o f the organ i sms . The d a t a p r e s e n t e d here shows t h a t A.cryptum has the c a p a c i t y t o b i n d to s u l p h u r c o n t a i n i n g m i n e r a l s and t h e r e f o r e would be i n a p o s i t i o n to enhance the growth o f T.ferrooxidans. A concern when comparing the B i o - R a d assay and the Ore ELISA was the d i f f e r e n c e i n r e q u i r e d c e l l s n e c e s s a r y f o r s a t u r a t i o n . A 10 f o l d i n c r e a s e i n c e l l s added t o the Ore ELISA over c e l l s added t o the B i o - R a d assay was n e c e s s a r y t o r e a c h an e q u i v a l e n t s a t u r a t i o n p o i n t . That i s , w h i l e 60% o f the c e l l s added to the B i o - R a d assay had bound t o the c h a l c o p y r i t e , o n l y 6% o f the c e l l s added were bound i n the Ore E L I S A . T h i s d i f f e r e n c e i n r e s u l t s i s l i k e l y due t o the s u b s t a n t i a l r e d u c t i o n i n m i n e r a l s u r f a c e a r e a 100 a v a i l a b l e f o r c e l l adherence i n the Ore ELISA as compared to the. Bio-Rad assay. Approximately 18 mg of mineral ore i s bound to each ELISA w e l l , of which a p o r t i o n of t h i s , t h a t i s i n v o l v e d w i t h the ore-glue i n t e r f a c e , would not have been a v a i l a b l e f o r c e l l attachment. When converted t o numbers of c e l l s bound per m i l l i g r a m of ore, the values recorded between assays were s i m i l a r . However the Bio-Rad assay u s u a l l y measured a s l i g h t l y higher number of bound c e l l s than the Ore ELISA. This v a r i a t i o n can be ex p l a i n e d by the inherent d i f f e r e n c e s between the assays. The Bio-Rad assay separates bound c e l l s from unbound c e l l s through d i f f e r e n t i a l c e n t r i f u g a t i o n . I t i s l i k e l y t h a t during c e n t r i f u g a t i o n , some unbound c e l l s as w e l l as weakly bound c e l l s are removed from suspension w i t h the p e l l e t i n g ore p a r t i c l e s . The Ore ELISA, w i t h i t s s e v e r a l washing steps, has greater stringency and detects only t e n a c i o u s l y bound c e l l s . As a r e s u l t , the Bio-Rad assay w i l l record a higher number of c e l l s bound. The Ore ELISA and Bio-Rad assay generated very s i m i l a r r e s u l t s on the k i n e t i c s of A.cryptum adherence to c h a l c o p y r i t e . Both b i n d i n g isotherms suggest t h a t each c e l l adheres independently to form a c e l l monolayer. Adherence occurs r a p i d l y and i s complete by 20 t o 25 min which compares w e l l w i t h data f o r T. ferrooxidans (Myerson & K l i n e , 1983; Tuovinen et a l . , 1983). 101 Studies have shown that the r e l a t i o n s h i p between pH and c e l l adherence depends on the s u b s t r a t e . Tuovinen (1983) and D i S p i r i t o (1983) found t h a t adherence to g l a s s beads increased w i t h decreasing pH but adsorption to mineral surfaces was independent, of pH. The l a t t e r i s comparable to the Ore ELISA r e s u l t s which showed l i t t l e r e l a t i o n s h i p between c h a l c o p y r i t e b i n d i n g and pH. The Bio-Rad assay r e s u l t s compared more c l o s e l y t o the g l a s s bead adsorption. These r e s u l t s could be e x p l a i n e d by the f a c t that the mineral i s the only surface exposed i n the Ore ELISA where as the mineral and the polypropylene microfuge tube w a l l are exposed to the c e l l i n the Bio-Rad assay. Therefore, attachment to polypropylene l i k e attachment to g l a s s beads, may be pH dependant. The e f f e c t of surface a c t i v e agents on i n c r e a s i n g the b i n d i n g of A.cryptum t o c h a l c o p y r i t e was s i m i l a r to that reported f o r T.ferrooxidans (Duncan et a l . , 1964; Kingma et a l . , 1979; Starkey, 1956). An explanation f o r t h i s observation i s t h a t the s u r f a c t a n t reduces the dynamic surface t e n s i o n enabling A.cryptum t o be more r e a d i l y adsorbed to the ore surface. In these s t u d i e s both A.cryptum and T.ferrooxidans gave s i m i l a r r e s u l t s . P r i o r to t h i s report the use of p u r i f i e d p r o t e i n s ( i e . BSA, g e l a t i n or SPP) i n a c i d o p h i l e adherence i n h i b i t i o n s t u d i e s had not been i n v e s t i g a t e d . Both BSA and g e l a t i n s t r o n g l y i n h i b i t c e l l adherence to c h a l c o p y r i t e . These p r o t e i n s probably act by 102 b l o c k i n g b i n d i n g s i t e s on the mineral surface. This a l s o appeared to be a g e n e r a l i z e d phenomenon o c c u r r i n g i n both A.cryptum and T. ferrooxidans attachment to ore. This f i n d i n g could have a serio u s impact on i n d u s t r i a l b i o l e a c h i n g processes. That i s , as the age and d e n s i t y of a c u l t u r e i n c r e a s e s , the concen t r a t i o n of secreted p r o t e i n s or other macromolecules w i l l i n c r e a s e . Therefore, i r o n i c a l l y , the c r u c i a l act of adherence may be i n h i b i t e d by macromolecules r e l e a s e d from A.cryptum and T. ferrooxidans. The i n h i b i t o r y e f f e c t s of BSA on c e l l adherence to surfaces i s not unique t o A.cryptum. For example, Gibbons (1972) has shown tha t the adherence of s e v e r a l species of o r a l b a c t e r i a to hydroxyapatite beads i s i n h i b i t e d by BSA. The exact mechanism by which A.cryptum attaches t o c h a l c o p y r i t e i s s t i l l u n clear. A l e c t i n - l i k e i n t e r a c t i o n was not detected given the l i m i t e d number of sugars i n v e s t i g a t e d . Supplemental st u d i e s on polysaccharide mediated adherence w i l l v e r i f y t h i s f i n d i n g . Many p u t a t i v e i n h i b i t o r s enhanced c e l l adherence. The increased adherence recorded w i t h monovalent c a t i o n s i s l i k e l y due t o the reduced l a t e r a l r e p u l s i v e forces experienced by adsorbing p a r t i c l e s i n concentrated e l e c t r o l y t e s o l u t i o n s (Rutter and Vincent, 1984). Di v a l e n t c a t i o n s and c h e l a t i n g agents probably act i n the same way i n causing e l e v a t e d c e l l adherence to c h a l c o p y r i t e . Both chemical agents can mediate c e l l aggregation by l i n k i n g charged groups on two b a c t e r i a l c e l l s u rfaces. 1 0 3 I t doesn't appear as though c e l l v i a b i l i t y i s necessary f o r c h a l c o p y r i t e adherence since c e l l adherence t o c h a l c o p y r i t e was maintained i n the presence of b a c t e r i c i d a l agents. Many researchers are s t i l l d i v i d e d on t h i s question (Beck, 1967; Tuovinen et a l . , 1983). Reducing agents appear to have l i t t l e e f f e c t on c e l l adherence. The e f f e c t of sulphur b i n d i n g agents i s i n c o n c l u s i v e and should be viewed w i t h caution due to the i n s t a b i l i t y of these agents at such a low pH. Hydrophobic i n t e r a c t i o n s are considered t o be an e s s e n t i a l aspect i n b a c t e r i a l adherence to. surfaces and i n t e r f a c e s (Marshall & Cruickshank, 1973; Dahlback et a l . , 1981). Stenstrom (1989) drew a c o r r e l a t i o n between c e l l surface hydrophobicity of v a r i o u s Salmonella typhimurium s t r a i n s and adherence to the minerals; quartz, a l b i t e , f e l d s p a r , and magnetite. From t h i s study the r o l e of hydrophobicity i n the attachment of A.cryptum t o mineral surfaces i s not c l e a r . Enhanced adherence was observed under c o n d i t i o n s of high i o n i c c o n c e n t r a t i o n or low pH (high proton concentration) suggesting t h a t hydrophobicity may pla y a r o l e i n mineral adherence. However, t h i s c o n c l u s i o n was not supported by our c e l l surface hydrophobicity s t u d i e s . That i s , w hile some adherence-defective s t r a i n s of A.cryptum demonstrated increased c e l l surface hydrophobicity, other adherence-defective s t r a i n s showed increased h y d r o p h i l i c i t y . 104 D i r e c t comparison of attachment of d i f f e r e n t b a c t e r i a l species was achieved through competitive i n h i b i t i o n • s t u d i e s . I t was shown th a t A.cryptum and T. ferrooxidans do not compete f o r attachment to c h a l c o p y r i t e . The r e s u l t s suggest that each b a c t e r i a l species has unique attachment s i t e s on the c h a l c o p y r i t e mineral surface. This f i n d i n g i s of great s i g n i f i c a n c e i n the study of m i c r o b i a l adherence to mineral surfaces. I t provides the f i r s t documented evidence to suggest that b a c t e r i a l attachment to mineral surfaces i s a s p e c i f i c process t h a t i s unique among d i f f e r e n t s t r a i n s of b a c t e r i a . In a d d i t i o n , the lack of competitive b i n d i n g between A.cryptum and T.ferrooxidans t o c h a l c o p y r i t e i m p l i e s t h a t the mechanisms of attachment f o r each c e l l type may d i f f e r . From an e c o l o g i c a l stand p o i n t , non-competitive attachment to mineral surfaces would b e n e f i t both A.cryptum and T.ferrooxidans. This would a l l o w the establishment of a c l o s e p h y s i c a l a s s o c i a t i o n between the two organisms necessary f o r c r o s s -feeding. In a d d i t i o n , the establishment and p r o l i f e r a t i o n of e i t h e r organism would not be r e s t r i c t e d by attachment s i t e c o n s t r a i n t s due to b i n d i n g competition between s t r a i n s . Several l i n e s of evidence suggest that a 31.6 kDa p r o t e i n may be i n v o l v e d i n the adherence of A.cryptum to c h a l c o p y r i t e . The 31.6 kDa p r o t e i n does not appear to be a s s o c i a t e d w i t h the c e l l s as t y p i c a l f i m b r i a e - l i k e s t r u c t u r e s . That i s , v i s i b l e surface 105 processes were not evident i n negative s t a i n e d micrographs or immunogold l a b e l l e d micrographs. Heat treatment or treatment wi t h reducing agents p r i o r to e l e c t r o p h o r e s i s d i d not generate detectable subunits of t h i s . p r o t e i n . The 31.6 kDa p r o t e i n appears t o be a s s o c i a t e d w i t h the outer membrane and can be removed from the surface of c e l l s by p h y s i c a l means. In a d d i t i o n , t h i s p r o t e i n may be at l e a s t , i n p a r t , hydrophobic i n nature. This was presumed because the p a r t i a l l y p u r i f i e d p r e p a r a t i o n (SPP) of t h i s p r o t e i n was impossible t o s o l u b i l i z e i n d i s t i l l e d water, and immunogold l a b e l l i n g i n d i c a t e d t h a t the p r o t e i n formed aggregates when dis l o d g e d from the c e l l s u r f a c e . Conclusive evidence v e r i f y i n g t h a t the 31.6 kDa p r o t e i n mediates mineral surface adherence i s s t i l l l a c k i n g . However, s e v e r a l observations were made which would suggest t h a t t h i s p r o t e i n i s i n v o l v e d i n attachment: Figure 14 and the antibody adsorption st u d i e s show t h a t t h i s i s the only p r o t e i n missing i n adherence-d e f e c t i v e mutants. A mutant showing enhanced ore adherence contained the 31.6 kDa p r o t e i n . The " f o o t p r i n t " l e f t by an adherent c e l l i n Figure 21 suggests t h a t the p r o t e i n may be i n v o l v e d i n attachment. This observation i s s i m i l a r t o what was described by M a r s h a l l (1971) who showed th a t mechanical shearing of b a c t e r i a from surfaces produced a polymer f o o t p r i n t . This argument i s f u r t h e r supported by the f a c t t h a t the only e f f e c t i v e i n h i b i t o r of c h a l c o p y r i t e b i n d i n g by A.cryptum was p r o t e i n . Convincing evidence t o demonstrate t h a t the 31.6 kDa p r o t e i n i s 106 d i r e c t l y i n v o l v e d i n c e l l adherence would be through i n h i b i t i o n s t u d i e s u s i n g p u r i f i e d p r o t e i n or monospecific antibody t o the p u t a t i v e adhesin. Both of these experiments were attempted w i t h l i m i t e d success due t o complications a r i s i n g from the type assays used. Further research i n t h i s area would help t o e l u c i d a t e the r o l e of the 31.6 kDa p r o t e i n i n c e l l adherence. Attempts t o i d e n t i f y the s t r u c t u r a l a s s o c i a t i o n of t h i s p r o t e i n w i t h the c e l l surface through c e l l treatment s t u d i e s were i n c o n c l u s i v e . I f the 31.6 kDa p r o t e i n i s i n v o l v e d i n mineral attachment, p r o t e i n a s e K or p h y s i c a l l y t r e a t e d c e l l s should have d i s p l a y e d reduced c h a l c o p y r i t e adherence. The r e s u l t s recorded, however, are not uncommon since many c e l l surface p r o t e i n s are i n a c c e s s i b l e or r e s i s t a n t t o exogenous enzymes. In a d d i t i o n , p h y s i c a l treatment of the c e l l s i s not l i k e l y to remove a l l surface components and as such may not e f f e c t c e l l adherence. For example, treatments which removed the haemagglutonin from Streptococcus sanguis c e l l surfaces d i d not reduce the b i n d i n g a b i l i t y of the t r e a t e d c e l l s (McBride, pers. comm.). I t should be noted t h a t mechanisms other than the 31.6 kDa p r o t e i n may be i n v o l v e d i n ore adherence. The i n a b i l i t y t o generate non-adherent mutants a f t e r exhaustive enrichments, suggests t h a t the other adherence f a c t o r s , necessary f o r c e l l v i a b i l i t y , may be i n v o l v e d . The l i m i t a t i o n s of the Bio-Rad assay prompted the development of 107 the Ore ELISA. The Ore ELISA can provide adherence measurements of s p e c i f i c c e l l types such t h a t competition f o r b i n d i n g s i t e s between two c e l l types, can be performed. The mineral s u b s t r a t e f o r adherence i s immobilized to the ELISA p l a t e , t h e r e f o r e , i n t e r f e r e n c e by mineral f i n e s i s e l i m i n a t e d . This assay i s p a r t i c u l a r l y adapted t o c h a r a c t e r i z a t i o n s t u d i e s : The p o t e n t i a l f o r m u l t i p l e runs of a given t e s t c o n d i t i o n , provides s t a t i s t i c a l l y s i g n i f i c a n t and r e l i a b l e r e s u l t s . A v a r i e t y of p o t e n t i a l i n h i b i t o r s can be screened w i t h l i t t l e a f f e c t on assay f i d e l i t y . When preparing the p l a t e s , various mineral substrates can be s u b s t i t u t e d f o r comparative a n a l y s i s . In a d d i t i o n , t h i s assay i s a powerful t o o l f o r screening adherence-defective mutants. An added fea t u r e of t h i s assay i s tha t i f c o l o u r development exceeds the det e c t a b l e A 4 0 5 range, the p l a t e s can be emptied, new subs t r a t e added and inc u b a t i o n shortened u n t i l d e s i r a b l e A 4 0 5 values can be read. In summary, t h i s study demonstrates t h a t A.cryptum adherence t o c h a l c o p y r i t e i s r a p i d , s a t u r a t i n g , and tenacious. Surface a c t i v e agents and c h e l a t i n g agents increased adherence. Bi n d i n g was i n h i b i t e d by a strong o x i d i z i n g agent and by p r o t e i n s , suggesting that reduced metals and polypeptides may be i n v o l v e d i n adherence. This was supported by st u d i e s which show the absence of a 31.6 kDa c e l l surface p r o t e i n on adherence-defective mutants. 108 BIBLIOGRAPHY 1. Adelberg, E.A., M. Mandel, and G.C.C. Chen. 1965. Optimal c o n d i t i o n s f o r mutagenesis by N-methyl-N-nitro-N-n i t r o s o g u a n i d i n e i n Escherichia coli K12. Biochem. Biophys. Res. Commun. 18:788. 2. Agate, A.D., and N.J. Khinvasara. 1985. B i o l e a c h i n g of copper ores and concentrate of Malanjkhand area, I n d i a , p. 83-90. In H.L. E h r l i c h and D.S. Holmes (ed.). Workshop on Biotechnology f o r the Mining, M e t a l - r e f i n i n g and Fo s s e l f u e l P rocessing I n d u s t r i e s . B i o t e c h n o l . Bioeng. Symp. Ser. 16. John Wiley and Sons. N.Y. 3. Alexander, B., S. Leach, and W.J. Ingledew. 1987. The r e l a t i o n s h i p between chemiosmotic parameters and s e n s i t i v i t y t o anions and organic acids i n the a c i d i p h i l e Thiobacillus ferrooxidans. J . Gen. M i c r o b i o l . 133:1171-1179. 4. Alexander, M. 1977. I n t r o d u c t i o n to S o i l M i c r o b i o l o g y , 2nd ed. Wiley, New York. 5. Andrews, and J . Maczuga, B i o t e c h n o l . Bioeng. Symp. Ser. 12, 337 (1982). 6. Andrews, G. 1988. The s e l e c t i v e adsorption of Thiobacilli t o d i s l o c a t i o n s i t e s on p y r i t i c s u r f a c e s . B i o t e c h n o l . Bioeng. 31:378-381. 7. Andrews, G., M. Darroch, and T. Hansson. 1988. B a c t e r i a l Removal of P y r i t e from Concentrated Coal S l u r r i e s . B i o t e c h n o l . Bioeng. 32:813-820. 8. Apel, W.A., P.R. Dugan, J.A. F i l p p i , and M.S. Rheins. 1976. Detection of Thiobacillus ferrooxidans i n a c i d mine environments by i n d i r e c t f l u o r e s c e n t antibody s t a i n i n g . Appl. Environ. M i c r o b i o l . 32:159-165. 9. Arkesteyn, G.J.M.W., and J.A.M. DeBont. 1980. T. acidophilus: a study of i t s presence on T. ferrooxidans c u l t u r e s . Can. J . M i c r o b i o l . 26:1057-1065. 10. Bagdigian, R.M., and A.S. Myerson. 1986. The adsorption of Thiobacillus ferrooxidans on co a l s u r f a c e s . B i o t e c h n o l . Bioeng. 28:467-479. 109 11. Baker, K.I., and A.L. M i l l s . 1982. Determination of the number of r e s p i r i n g Thiobacillus ferrooxidans c e l l s i n water samples by u s i n g combined f l u o r e s c e n t a n t i b o d y - 2 - ( p - I o d o p h e n y l ) - 3 - ( p - N i t r o p h e n y 1 ) - 5 -P h e n y l t e t r a z o l i u m c h l o r i d e s t a i n i n g . Appl. Environ. M i c r o b i o l . 43:338-344. 12. Baldensperger. J . , L.J. Guarraia, and W.J. Humphreys. 1974. Scanning e l e c t r o n microscopy of Thiobacilli grown on c o l l o i d a l s u l f u r . Arch. M i c r o b i o l . 99:323-329. 13. Beachey, E.H. 1981. B a c t e r i a l adherence: adhesin-receptor i n t e r a c t i o n s mediating the attachment of b a c t e r i a to mucosal su r f a c e s . J . I n f e c t . D i s . 143:325-345. 14. Beck, J.V., and D.G. Brown. 1968. D i r e c t s u l f i d e o x i d a t i o n i n the s o l u b i l i z a t i o n of s u l f i d e ores by T.ferrooxidans. J . B a c t e r i o l . 96:1433-1434. 15. Beck, J.V. 1967. The r o l e of b a c t e r i a i n copper mining operations. B i o t e c h n o l . Bioeng. 9:487-497. 16. Bennett, J.C., and H. T r i b u t s c h . 1978. B a c t e r i a l l e a c h i n g p a t t e r n s on p y r i t e c r y s t a l surfaces. J . B a c t e r i o l . 134:310-317. 17. Berry, V.K., and L.E. Murr. 1975. B a c t e r i a l attachment to molybdenite: An e l e c t r o n microscope study. M e t a l l . Trans. B6:488. 18. Berry, V.K., and L.E. Murr. 1978. D i r e c t observation of b a c t e r i a and q u a n t i t a t i v e s t u d i e s of t h e i r c a t a l y t i c r o l e i n the l e a c h i n g of low-grade, copper-bearing waste, p. 103-136. In L.E. Murr, A.E. Torma and J.A. B r i e r l e y (ed.), M e t a l l u r g i c a l A p p l i c a t i o n s of B a c t e r i a l Leaching and r e l a t e d M i c r o b i o l o g i c a l Phenomena. Academic Press. N.Y. 19. Boateng, D.A.D., and C.R. P h i l l i p s . 1977. D e s u l f u r i z a t i o n of c o a l by f l o a t a t i o n of c o a l i n a s i n g l e - s t a g e process. Sep. S c i . 12:71-86. 20. Boyd, J , and B.C. McBride. 1984. F r a c t i o n a t i o n of hemagglutinating and b a c t e r i a l b i n d i n g adhesins of B a c t e r i o d e s gingivalis. I n f e c t . Immun. 45:403-409. 21. B r i e r l e y , C.L., J.A. B r i e r l e y , and L.E. Murr. 1973. Using the SEM i n mining research. Res. Dev. 24:24. 110 22. B r i e r l e y , C.L., and L.E. Murr. 1973. Leaching: Use of a t h e r m o p h i l i c and chemoautotrophic microbe. Science. 179:488. 23. B r i e r l e y , C L . 1977. Thermophilic microorganisms i n the e x t r a c t i o n of metals from ores. Dev. Ind. M i c r o b i o l . 18:273-284. 24. B r i e r l e y , C L . 1978. B a c t e r i a l Leaching. CRC C r i t . Revs. M i c r o b i o l . 6:207-262. 25. Brock, T.D. 1979. Biology of Microorganisms. P r e n t i c e -H a l l , Inc., Englewood C l i f f s , New Jersey. 26. Bryant, R.D., J.W. Costerton, and E.J. L a i s h l e y . 1984. The r o l e of T.albertis g l y c o c a l y x i n the adhesion of c e l l s to elemental s u l f u r . Can. J . M i c r o b i o l . 30:81-90. 27. Campbell, R.S., and O.T. L i n d . 1969. Water q u a l i t y and aging of s t r i p mine l a k e s . J . Water P o l l u t . Contr. Fed. 41:1943-1955. 28. Carpenter, K.E. 1925. On the b i o l o g i c a l f a c t o r s i n v o l v e d i n the d e s t r u c t i o n of r i v e r f i s h e r i e s by p o l l u t i o n due t o lead-mining. Ann. Appl. B i o l . 12:1-13. 29. Chang, Y.C, and A.S. Myerson. 1982. Growth models of the continuous b a c t e r i a l l e a c h i n g of i r o n p y r i t e by T. ferrooxidans. B i o t e c h , and Bioeng. 24:889-902. 30. C i s a r , J.O., S.H. C u r l , P.E. Kolenbrander, and A.E. V a t t e r . 1983. S p e c i f i c absence of type 2 fimbriae on a coaggregation-defective mutant of Actinomyces viscosus T14V. I n f e c t . Immun. 40:759-765. 31. C i s a r , J.O., A.L. Sandberg, and S.E. Mergenhagen. 1984. The f u n c t i o n and d i s t r i b u t i o n of d i f f e r e n t fimbriae on s t r a i n s of Actinomyces viscosus and Actinomyces maeslundii. J . dent. Res. 63:393-396. 32. Costerton, J.W., and R.T. I r v i n . 1981. The b a c t e r i a l g l y c o c a l y x i n nature and disease. Annu. Rev. M i c r o b i o l . 351-299-324. 33. Dahlback, B., M. Hermansson, S. K j e l l e b e r g , and B. Norkrans. 1981. The hydrophobicity of b a c t e r i a - an important f a c t o r i n t h e i r i n i t i a l adhesion at a i r -water i n t e r f a c e . Arch. M i c r o b i o l . 128:267-270. 34. Dazzo, F.B., and W.J. B r i l l . 1978. Reg u l a t i o n by f i x e d n i t r o g e n of host-symbiont r e c o g n i t i o n i n the Rhizobium-clover symbiosis. P l a n t P h y s i o l . 62:18-21. I l l 35. Dazzo, F.B., G.L. Truchet, J.E. Sherwood, E.H. Hrabak, and A.E. G a r d i o l . 1982. A l t e r a t i o n of the t r i f o l i i n A-bi n d i n g capsule of Rhizobium t r i f o l i i 0403 by enzymes r e l e a s e d from c l o v e r r o o t s . Appl. Environ. M i c r o b i o l . 44:478-490. 36. Dazzo, F.B., and G.L. Truchet. 1983. I n t e r a c t i o n s of l e c t i n s and t h e i r saccharide receptors i n the Rhizobium-legume symbiosis. J . Membr. B i o l . 73:1-16. 37. Detz, CM., and G. Barvinchak. 1979. M i c r o b i a l d e s u l f u r i z a t i o n of c o a l . Mining Congress J . 86:78-83. 38. D i S p i r i t o , A.A., M. S i l v e r , L. Voss, and O.H. Tuovinen. 1981. F l a g e l l a and p i l i of i r o n o x i d i z i n g t h i o b a c i l l i i s o l a t e d from a uranium mine i n Northern Ontario, Canada. Appl. Environ. M i c r o b i o l . 27:850-853. 39. D i S p i r i t o , A.A., P.R. Dugan, and O.H. Tuovinen. 1983. Sor p t i o n of T.ferrooxidans to p a r t i c u l a t e m a t e r i a l . B i o t e c h n o l . Bioeng. 25:1163-1168. 40. Doyle, R.J., W.E. N e s b i t t , and K.G. Taylor. 1982. On the mechanism of adherence of Streptococcus sanguis t o hydroxyapatite. FEMS M i c r o b i a l . L e t t . 15:1-5. 41. Dugan, P.R., and W.A. Apel. 1978. M i c r o b i a l d e s u l f u r i z a t i o n of c o a l . I_n L.E. Murr, A.E. Torma and J.A. B r i e r l e y (ed.), M e t a l l u r g i c a l A p p l i c a t i o n s of B a c t e r i a l Leaching and Related M i c r o b i o l o g i c a l Phenomena. Academic Press Inc. (N.Y.) 1978. 42. Dugan, P.R. 1986. M i c r o b i o l o g i c a l d e s u l f u r i z a t i o n of co a l and i t s increased monetary value, p. 185-221. Tn H.L. E h r l i c h and D.S. Holmes (ed.), Workshop on Biotechnology f o r the Mining, M e t a l - R e f i n i n g and F o s s i l Fuel Processing i n d u s t r i e s . B i o t e c h . and Bioeng. Symp. No. 16. John Wiley and Sons. New York. 43. Duncan, D.W., P.C. T r u s s e l l , and C C . Walden. 1964. Leaching of c h a l c o p y r i t e w i t h Thiobacillus ferrooxidans. E f f e c t of s u r f a c t a n t s and shaking. Appl. M i c r o b i o l . 12:122-126. 44. Duncan, D.W., and A.D. Drummond. 1973. M i c r o b i o l o g i c a l l e a c h i n g of porphyry copper type m i n e r a l i z a t i o n : P o s t - l e a c h i n g observations. Can. J . Ear t h S c i . 10:476. 112 45. E n g v a l l , E., and P. Perlmann. 1972. Enzyme-linked immune-adsorbent assay, ELISA. J . Immunol. 109:129-135. 46. Espejo, R.T., and P. Romero. 1987. Growth of T h i o b a c i l l u s f e r r o o x i d a n s on elemental s u l f u r . Appl. Environ. M i c r o b i o l . 52:1907-1912. 47. Fachon-Kalweit, S., B.L. El d e r , and P. F i v e s - T a y l o r . 1985. A n t i b o d i e s that b i n d to fi m b r i a e block adhesion of Streptococcus sanguis t o s a l i v a - c o a t e d hydroxyapatite. I n f e c t . Immun. 48:617-624. 48. F i v e s - T a y l o r , P.M., and D.W. Thompson. 1985. Surface p r o p e r t i e s of Streptococcus sanguis FW213 mutants nonadherent t o s a l i v a - c o a t e d hydroxyapatite. I n f e c t . Immun. 47:752-759. 49. F l e t c h e r , M., M.J. Latham, J.M. Lynch, and P.R. Rut t e r . 1980. The c h a r a c t e r i s t i c s of i n t e r f a c e s and t h e i r r o l e i n M i c r o b i a l attachment, p. 67-78. In R.C.W. Berkeley, J.M. Lynch, J . M e l l i n g , P.R. Rut t e r , and B. Vincent (ed.), M i c r o b i a l Adhesion t o su r f a c e s . Horwood, Chichester. 50. Gaastra, W., and F.K. de Gra f f . 1982. H o s t - s p e c i f i c f i m b r i a l adhesins of noninvasive e n t e r o t o x i g e n i c E s c h e r i c h i a coli s t r a i n s . M i c r o b i o l . Rev. 46:129-161. 51. Ganeshkumar, N. M.Sc. t h e s i s . October 1985. U.B.C. 52. Gibbons, R.J., and J . van Houte, and W.F. L i l j e m a r k . 1972. Parameters that a f f e c t the adherence of Streptococcus salivarius to o r a l e p i t h e l i a l s u r f a c e s . J . Dent. Res. 51:424-435. 53. Gonzalez, C , and D. Cotoras. 1987. Apendices de T h i o b a c i l l u s ferrooxidans en c u l t i v o s sucesivos. R e v i s t a Latinoamerica de M i c r o b i o l o g i a 29:235-238. 54. Gormely, L.S., and D.W. Duncan. 1974. E s t i m a t i o n of T. f e r r o o x i d a n s concentrations. Can. J . M i c r o b i o l . 20:1453-1455. 55. Gormely, L.S., D.W. Duncan, R.M.R. Branion, and K.L. Pinde r . 1975. Continuous c u l t u r e of T. f e r r o o x i d a n s on a z i n c s u l f i d e concentrate. B i o t e c h . Bioeng. 27:31-49. 113 56. Handley, P.S., P.L. Carter, J.E. Wyatt, and L.M. Hesketh. 1985. Surface s t r u c t u r e s ( P e r i t r i c h o u s f i b r i l s and t u f t s of f i b r i l s ) found on Streptococcus sanguis s t r a i n s may be r e l a t e d to t h e i r a b i l i t y to coaggregate wi t h other o r a l genera. I n f e c t . Immun. 47:217-227. 57. H a r r i s o n , A.P., B. J a r v i s , and J.L. Johnson. 1980. H e t e r o t r o p h i c b a c t e r i a from c u l t u r e s of A u t o t r o p h i c Thiobacillus ferrooxidans: R e l a t i o n s h i p s as s t u d i e d by means of d e o x y r i b o n u c l i c a c i d homology. J . B a c t e r i o l . 143:448-454. 58. H a r r i s o n , A.P. 1981. Acidophilium cryptum gen. nov., sp. nov. Heterotrophic b a c t e r i a from a c i d i c mineral environments. I n t . J . Syst. B a c t e r i o l . 31:327-332. 59. H a r r i s o n , A.P. 1982. Genomic and p h y s i o l o g i c a l d i v e r s i t y amongst s t r a i n s of T. ferrooxidans and genomic comparison w i t h T.thiooxidans. Arch. M i c r o b i o l . 131:68-76. 60. Heckels, J.E., B. B l a c k e t t , J.S. Everson, and M.E. Ward. 1976. The i n f l u e n c e of surface charge on the attachment of N e i s s e r i a gonorrhoae to human c e l l s . J . Gen. M i c r o b i o l . 96:359-364. 61. Hermansson, M., S. K j e l l e b e r g , T.K. Korhonen, and T.A. Stenstrom. 1982. Hydrophobic and e l e c t r o s t a t i c c h a r a c t e r i z a t i o n of surface s t r u c t u r e s of b a c t e r i a and i t s r e l a t i o n s h i p to adhesion at an a i r - w a t e r s u r f a c e . Arch. M i c r o b i o l . 131:308-312. 62. H i l t u n e n , P., V. Vuorinen, P. R e h i t i j a r v i and O.T. Tuovinen. 1981. B a c t e r i a l p y r i t e o x i d a t i o n : Release of i r o n and scanning e l e c t r o n m i c r o s c o p i c o b s e r v a t i o n s . Hydrometallurgy, 7:147-157. 63. Hoffman, M.R., B.C. Faust, F.A. Panda, F.A. Koo, H.H. and H.M. Tsuchiya. 1981. K i n e t i c s of the removal of i r o n p y r i t e from c o a l by m i c r o b i a l c a t a l y s i s . Appl. Environ, M i c r o b i o l . , 42: 259-271. 64. Hoppe, H.G. 1984. Attachment of b a c t e r i a : Advantage or disadvantage f o r s u r v i v a l i n the aquatic environment, p. 283-301. In K.C. M a r s h a l l (ed.), M i c r o b i a l Adhesion and Aggregation. S p r i n g e r - V e r l a g , B e r l i n . 114 6 5 . Hrabak, E.M., M.R. Urbano, and F.B. Dazzo. 1 9 8 1 . Growth-phase dependent immunodeterminants of Rhizobium t r i f o l i i l i p o p o l y s a c c h a r i d e which bind t r i f o l i i A, a white c l o v e r l e c t i n . J . B a c t e r i o l . 1 4 8 : 6 9 7 - 7 1 1 . 6 6 . Huber, T.F., N.W.F. Kossen, P.Bos and J.G. Kuenen. 1 9 8 4 . Design and scale, up of a reac t o r f o r m i c r o b i a l d e s u l p h u r i z a t i o n of c o a l . pp. 1 7 9 - 1 9 0 . In E.H. Houwink and R.R. van der Meer (ed.), Innovations of Biotechnology: Progress i n i n d u s t r i a l M i c r o b i o l o g y , V o l . 2 0 . E l s e v i e r Science P u b l i s h i n g Co. Inc. (N.Y.) . 6 7 . Ingledew, W.J., and J.G. Cobley. 1 9 8 0 . A p o t e n t i o m e t r i c and k i n e t i c study on the r e s p i r a t o r y chain of ferro u s grown T. ferrooxidans. Biochem. Biophys. Acta. 5 9 0 : 1 4 1 - 1 5 8 . 6 8 . Ingledew, W.J. 1 9 8 2 . Thiobacillus ferrooxidans. The bi o e n e r g e t i c s of an a c i d o p h i l i c chemolithotroph. Biochimica et Biophysica acta. 6 8 3 : 8 9 - 1 1 7 . 6 9 . Isaacson, R.E. 1 9 8 5 . P i l u s adhesions, p. 3 0 7 - 3 3 6 . In D.C. Savage and M. F l e t c h e r (ed.), B a c t e r i a l Adhesion: Mechanisms and P h y s i o l o g i c a l S i g n i f i c a n c e . Plenum Press. (N.Y.). 7 0 . Johnson, D.B., W.I. Kelos, and D.A. Jenkins. 1 9 7 8 . B a c t e r i a l streamer growth i n a disused p y r i t e mine. Environ. P o l l u t . 1 8 : 1 0 7 - 1 1 8 . 7 1 . Johnson, D.B., and W.I. Kelso. 1 9 8 3 . D e t e c t i o n of he t e r o t r o p h i c contaminants i n c u l t u r e s of T. ferrooxidans and t h e i r e l i m i n a t i o n by s u b c u l t u r i n g i n media c o n t a i n i n g CuSO,. J . Gen. M i c r o b i o l . 1 2 9 : 2 9 6 9 - 2 9 7 2 . 7 2 . Jones, G.E., and R.L. Starkey. 1 9 6 1 . Surface a c t i v e substances produced by Thiobacillus ferrooxidans. B a c t e r i o l . 8 2 : 7 8 8 - 7 8 9 . 7 3 . Jones, G.W., and R.E. Isaacson. 1 9 8 3 . P r o t e i n a c i o u s b a c t e r i a l adhesins and t h e i r receptors. C r i t . Rev. M i c r o b i o l . 1 0 : 2 2 9 - 2 6 0 . 7 4 . Karamanev, D.G., and L.N. Nikolov. 1 9 8 8 . I n f l u e n c e of some physicochemical parameters on b a c t e r i a l a c t i v i t y of b i o f i l m : Ferrous i r o n o x i d a t i o n by Thiobacillus ferrooxidans. B i o t e c h n o l . Bioeng. 3 1 : 2 9 5 - 2 9 9 . 115 75. K e l l e r , L., and L.E. Murr. 1982. A c i d - b a c t e r i a l and f e r r i c s u l f a t e leaching of p y r i t e s i n g l e c r y s t a l s . B i o t e c h . Bioeng. 24:83-96. 76. K e l l y , D.P., and C A . Jones. 1978. Fac t o r s a f f e c t i n g metobolism and ferrous i o n o x i d a t i o n i n suspensions and b a t c h c u l t u r e s of T. ferrooxidans. p. 19-45. In L.E. Murr, A. E. Torma, and J.A. B r i e r l e y (ed.), M e t a l l u r g i c a l A p p l i c a t i o n s of B a c t e r i a l Leaching and Related M i c r o b i o l o g i c a l Phenomena. Academic P r e s s , New York. 77. K e l l y , D.P., P.R. N o r r i s and C.L. B r i e r l e y . 1979. M i c r o b i o l o g i c a l methods f o r the e x t r a c t i o n and recovery of metals, p. 263-308. In A.T. B u l l , D.C. Ellwood and C. Ratledge (ed.), M i c r o b i a l Technology: Current s t a t e , future prospects. Cambridge U n i v e r s i t y P ress, Cambridge. 78. Kempton, A.G., R.G.L. McCready, and C.E. Capes. 1980. Removal of p y r i t e from coal by c o n d i t i o n i n g w i t h T h i o b a c i l l u s ferrooxidans followed by o i l agglomeration. Hydrometallurgy. 5:117-125. 79. K h a i l o v , K.M., and Z.Z. Fineko. 1970. Organic macromolecular compounds d i s s o l v e d i n sea-water and t h e i r i n c l u s i o n i n t o food chains, p. 6-18. Ln J.H. S t e e l e (ed.), Food Chains. Edinburgh: O l i v e r and Boyd. 80. Kingma J r . , J.G., and M. S i l v e r . 1979. A u t o t r o p h i c growth o f T h i o b a c i l l u s acidophilus i n the presence of a s u r f a c e - a c t i v e agent, Tween 80. Ap p l . . E n v i r o n . M i c r o b i o l . 38:795-799. 81. Kishimoto, N., and T. Tano. 1987. A c i d o p h i l i c heterotrophic bacteria i s o l a t e d from a c i d i c mine drainage, sewage and s o i l s . J. Gen. Appl. Microbiol. 33:11-25. 82. Kjelleberg, S. 1984. Adhesion to inanimate surfaces, p. 51-70. In K.C. Marshall (ed.), Mircrobial Adhesion and Aggregation. Springer-Verlag, B e r l i n . 83. Lacey, D.T., and F. Lawson. 1970. Kinetics of the l i q u i d -phase oxidation of acid ferrous s u l f a t e by the bacterium T h i o b a c i l l u s ferrooxidans. Biotechnol. Bioeng. 12:29. 84. Laemmli, U.K. 1970. Cleavage of st r u c t u r a l proteins during the assembly of the head of bacteriophage T4. Nature (London). 227:680-685. 116 85. Leech, R., and R.J.W. Hefford. 1980. The observation of b a c t e r i a l d e p o s i t i o n from a flo w i n g suspension, p. 544-545. In R.C.VI. Berkeley, J.M. Lynch, J . M e l l i n g , P.R. R u t t e r , and B. Vincent (ed.), M i c r o b i a l Adhesion t o Surfaces. Horwood, Chichester. 86. Lobos, J.H., T.E. Chisolm, L.H. Bopp, and D.S. Holmes. 1986. A c i d i p h i l i u m organovorum sp. nov., an a c i d i p h i l i c heterotroph i s o l a t e d from a Thiobacillus f e r r o o x i d a n s c u l t u r e . I n t . J . Syst. B a c t e r i o l . 36:139-144. 87. Lundgren, D.G., and T. Tano. 1978. S t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s of Thiobacillus r e l a t i v e t o f e r r o u s i r o n and s u l f i d e o x i d a t i o n s , p. 151-166. In L.E. Murr, A.E. Torma, and J.A. B r i e r l y (ed.), M e t a l l u r g i c a l A p p l i c a t i o n s of B a c t e r i a l Leaching and Related M i c r o b i o l o g i c a l Phenomena. Academic Press, New York. 88. Lundgren, D.G., and M. S i l v e r . 1980. Ore l e a c h i n g by b a c t e r i a . Ann. Rev. M i c r o b i o l . 34:263-283. 89. Manning, H.L. 1975. New medium f o r i s o l a t i n g i r o n -o x i d i z i n g and he t e r o t r o p h i c a c i d o p h i l i c b a c t e r i a from a c i d mine drainage. Appl. M i c r o b i o l . 30:1010-1016. 90. M a r s h a l l , K.C, R. Stout, and R. M i t c h e l l . 1971. Mechanism of the i n i t i a l events i n the s o r p t i o n of marine b a c t e r i a t o surfaces. J . Gen. M i c r o b i o l . 68:337-348. 91. M a r s h a l l , K.C, and R.H. Cruickshank. 1973. C e l l s u r f a c e h y d r o p h o b i c i t y and the o r i e n t a t i o n of c e r t a i n b a c t e r i a at i n t e r f a c e s . Arch. M i c r o b i o l . 91:29-40. 92. M a r s h a l l , K.C. 1980. B a c t e r i a l adhesion i n n a t u r a l environments, p. 187-238. In R.C.W. Berkeley, J.M. Lynch, J . M e l l i n g , P.R. Rutter and B. Vincent (ed.), M i c r o b i a l Adhesion to Surfaces. Horwood, C h i c h e s t e r . 93. M a r s h a l l , K.C. 1985. Mechanisms of b a c t e r i a l adhesion at so l i d - w a t e r i n t e r f a c e s , p. 133-161. In D.C. Savage and M. F l e t c h e r (ed)., B a c t e r i a l Adhesion: Mechanisms and P h y s i o l o g i c a l S i g n i f i c a n c e . Plenum Press. (N.Y.). 94. McBride, B.C., and M.T. Gisslow. 1977. Role of s i a l i c a c i d i n s a l i v a - i n d u c e d aggregation of Streptococcus sanguis. I n f e c t . Immun. 18:35-40. 117 McCready, R.G.L., and B.P. L e G a l l a i s . 1984. S e l e c t i v e a d s o r p t i o n of phosphorous-32-labelled T. ferrooxidans i n f i n e l y ground coal suspensions. Hydrometallurgy. 12:281-288. McGoran, C.J.M., D.W. Duncan, and C C . Walden. 1969. Growth of Thiobacillus ferrooxidans on v a r i o u s s u b s t r a t e s . Can. J . M i c r o b i o l . 15:135-138. Merker, R.I., and J . Smit. 1988. C h a r a c t e r i z a t i o n of the adhesive h o l d f a s t of marine and freshwater cauloJbacters. Appl. Environ. M i c r o b i o l . 54:2078-2085. M i l l s , A.L. 1985. A c i d Mine Waste Drainage: M i c r o b i a l Impact on the Recovery of S o i l and Water Ecosystems, p. 35-81. In R.L. Tate and D.A. K l e i n (ed.), S o i l Reclamation Processes: M i c r o b i o l o g i c a l Analyses and A p p l i c a t i o n s . Marcel Dekker, Inc. New York. M i l l s , A.L., and L.M. Mallory. 1987. The community s t r u c t u r e of s e s s i l e h e t e r o t r o p h i c b a c t e r i a s t r e s s e d by a c i d mine drainage. Microb. E c o l . 14:219-232. Murr, L.E., and V.K. Berry. 1976. Direct observation of s e l e c t i v e attachments of bacteria on low-grade s u l f u r ores and other mineral surfaces. Hydrometallurgy. 2:11-24. Muyzer, G., A.C deBruyn, D.J.M. Schmedding, P. Bos, P. Westbroek, and G.J. Kuenen. 1987. A combined i m m u n o f l u o r e s c e n c e - D N A - f l u o r e s c e n c e s t a i n i n g technique f o r enumeration of T h i o b a c i l l u s ferrooxidans i n a population of a c i d o p h i l i c b a c t e r i a . A p p l . Environ. M i c r o b i o l . 53:660-664. Myerson, A.S., and P. K l i n e . 1983. The a d s o r p t i o n of T h i o b a c i l l u s ferrooxidans on s o l i d s u r f a c e s . B i o t e c h n o l . Bioeng. 25:1669-167 6. N e s b i t t , W.E., R.J. Doyle, K.G. Taylor, R.S. S t a a t , and R.R. A r n o l d . 1982. P o s i t i v e c o o p e r a t i v i t y i n the binding of Streptococcus sanguis t o h y d r o x y a p a t i t e . I n f e c t . Immun. 35:157-165. N o r r i s , P.R., and D.P. K e l l y . 1978. D i s s o l u t i o n of p y r i t e by pure and mixed c u l t u r e s of some a c i d o p h i l i c b a c t e r i a . FEMS Microb. L e t t . 4:143-146. 118 105. N o r r i s , R.H., P.S. Lake, and R. Swain. 1981. E c o l o g i c a l e f f e c t s of mine e f f l u e n t s on the South Esk R i v e r , North-eastern Tasmania. I. Study area and b a s i c water c h a r a c t e r i s t i c s . Aust. J . Fresh water Res. 31:817-827. 106. Oakley, B.R., D.R. K i r s c h , and N.R. M o r r i s . 1980. A s i m p l i f i e d u l t r a s e n s i t i v e s i l v e r s t a i n f o r d e t e c t i n g p r o t e i n s i n polyacrylamide g e l s . Anal. Biochem. 105:361-363. 107. Olem, H. 1981. Coal and c o a l mine drainage. J . Water P o l l u t . Contr. Fed. 53:814-824. 108. Orskov, I . , F. Orskov, H.W. Smith, and W.J. Sojka. 1975. The establishment of K99, a t h e r m o l a b i l e , t r a n s m i s s i b l e E s c h e r i c h i a coli K antigen, p r e v i o u s l y c a l l e d "Kco", possessed by c a l f and lamb enteropathogenic s t r a i n s . Acta. P a t h o l . M i c r o b i o l . Scand. Sect. B. 83:31-36. 109. Orskov, I . , F. Orskov, W.J. Sojka, and J.M. Leach. 1961. Simultaneous occurrence of E.coli B and L antigens i n s t r a i n s from diseased swine. Acta. P a t h o l . M i c r o b i o l . Scand. Sect. B. 53:404-422. 110. Rao, G.S., and L.R. Berger. 1970. Basis of pyruvate i n h i b i t i o n of T h i o b a c i l l u s thiooxidans. J . B a c t e r i o l . 102:462-466. 111. Robb, I.D. 1984. Stereo-biochemistry and f u n c t i o n of polymers. p. 39-49. In K.C. Ma r s h a l l (ed.), M i c r o b i a l Adhesion and Aggregation. Springer-Verlag, B e r l i n . 112. R o l l a , G. 1976. I n h i b i t i o n of adsorption-general c o n s i d e r a t i o n s , p. 309-324. In H.M. S t i l e s , W.J. Loesche, and T.C. O'Brien (ed.), Proceedings: M i c r o b i a l Aspects of Dental Caries (a s p e c i a l supplement to Micr o b i o l o g y A b s t r a c t s ) , v o l . 2. Information R e t r i e v a l Inc., Washington, D.C. 113. Rosenberg, E., N. Kaplan, D. Pines, M. Rosenberg, and d. Gutnik. 1983(a). Capsular polysaccharides i n t e r f a c e w i t h adherence of A c i n e t o b a c t e r calcoaceticus to hydrocarbon. FEMS M i c r o b i o l . L e t t . 17:157-160. 114. Rosenberg, M., D. Gutnick, and E. Rosenberg. 1980. Adherence of b a c t e r i a to hydrocarbons: a simple method f o r measuring c e l l - s u r f a c e h y d r o p h o b i c i t y . FEMS M i c r o b i o l . L e t t . 9:29-33. 119 115. Rosenberg, M., E. Rosenberg, H. Judes, and E. Weiss. 1983(b). Hypothesis. B a c t e r i a l adherence t o hydrocarbons and t o surfaces i n the o r a l c a v i t y . FEMS M i c r o b i o l . L e t t . 20:1-5. 116. Roy, P., and A.K. Mishra. 1981. Factors a f f e c t i n g o x i d a t i o n of p y r i t e by T.ferrooxidans. Ind. J . of Exp. B i o l . 19:728-732. 117. Rutter, P.R., and B. Vincent. 1984. Physicochemical i n t e r a c t i o n s of the substratum, microorganism and the f l u i d - p h a s e , p. 21-38. In K.C. M a r s h a l l (ed.), M i c r o b i a l Adhesion and Aggregation. Springer-Verlag, B e r l i n . 118. Schaeffer, W.I., P.E. Holbe r t , and W.W. Umbreit. 1963. Attachment of Thiobacillus ferrooxidans t o s u l f u r c r y s t a l s . J . B a c t e r i o l . 85:137-142. 119. Schaeffer, W.L., and W.W. Umbreit. 1963. Ph o s p h o t i d y l i n o -s i t o l as a wetting agent i n s u l f u r o x i d a t i o n by T . thiooxidans. J . B a c t e r i o l . 85:492-493. 120. Schnaitman, C , and D.G. Lundren. 1965. Organic compounds i n the spent medium of Ferrobacillus ferrooxidans. Can. J . M i c r o b i o l . 11:23-27. 121. Schrader, J.A., and D.S. Holmes. 1988. Phenotypic s w i t c h i n g of Thiobacillus ferrooxidans. J . B a c t e r i o l . 170:3915-3923. 122. S h i w e r s , D.W., and T.D. Brock. 1973. Oxidation of elemental s u l f u r by Sulfolobus acidocaldarius. J . B a c t e r i o l . 114:706. 123. Shuttleworth, K.L., R.F. Uuz, and P.L. Wichlacz. 1985. Glucose catabolism i n s t r a i n s of a c i d o p h i l i c , h e t e r o t r o p h i c b a c t e r i a . Appl. Environ. M i c r o b i o l . 50:573-579. 124. Silverman, M.P. 1967. Mechanism of B a c t e r i a l p y r i t e o x i d a t i o n . J . B a c t e r i o l . 94:1046-1051. 125. Singer, P.C., and W. Stumm. 1970. A c i d i c mine drainage: The rate-determining step. Science. 167:1121-1126. 126. Staat, R.H., and J.C. Peyton. 1984. Adherence of o r a l s t r e p t o c o c c i : Evidence of n o n - s p e c i f i c adsorption to s a l i v a - c o a t e d hydroxyapatite surfaces. I n f e c t . Immun. 44:653-659. 120 127. Starkey, R.L., G.E. Jones, and L.R. F r e d e r i c k . 1956. E f f e c t s of medium a g i t a t i o n and wetting agents on o x i d a t i o n of sulphur by Thiobacillus thiooxidans. J . Gen. M i c r o b i o l . 15:329-334. 128. Stenstrom, T.A. 1989. B a c t e r i a l hydrophobicity, an o v e r a l l parameter f o r the measurement of adhesion p o t e n t i a l to s o i l p a r t i c l e s . Appl. Environ. M i c r o b i o l . 55:142-147. 129. S t e r r i t t , R.M., and J.N. L e s t e r . 1980. I n t e r a c t i o n s of heavy metals w i t h b a c t e r i a . S c i . Tot. Environ. 14:507. 130. Stotzky, G. 1985. Mechanisms of adhesions t o c l a y s , w i t h reference to s o i l systems, p. 195-253. Ln D.C. Savage and M. F l e t c h e r (ed.), B a c t e r i a l Adhesion: Mechanisms of P h y s i o l o g i c a l S i g n i f i c a n c e . Plenum Press. (N.Y.). 131. Sutherland, I.W. 1983. M i c r o b i a l exopolysaccharides t h e i r r o l e i n m i c r o b i a l adhesion i n aqueous systems. C r i t . Rev. M i c r o b i o l . 10:173-201. 132. Takakuwa, S., T. F u j i m o r i , and H. Iwasaki. 1979. Some p r o p e r t i e s of c e l l - s u l f u r adhesion i n T. thiooxidans. J . Gen. Appl. M i c r o b i o l . 25:21-29. 133. Torma, A.E., G.G. Gabra, R.Guay, and M. S i l v e r . 1976(a). E f f e c t of surface a c t i v e agents on the o x i d a t i o n of c h a l c o p y r i t e by T.ferrooxidans. Hydrometallurgy. 1:301-309. 134. Torma, A.E., and I . J . I t z k o v i t c h . 1976(b). Influence of organic solvents on c h a l c o p y r i t e o x i d a t i o n a b i l i t y of T. ferrooxidans. Appl. Environ. M i c r o b i o l . 32:102-107. 135. Torma, A.E. 1977. The r o l e of Thiobacillus ferrooxidans i n h y d r o m e t a l l u r g i c a l processes. Adv. Biochem. Eng. 6:1-37. 136. Torma, A.E., and K. Bosecker. 1982. B a c t e r i a l Leaching p. 77-118. In M.J. B a l l (ed.), Progress i n I n d u s t r i a l M i c r o b i o l o g y . V o l . 16. E l s e v i e r S c i e n t i f i c P u b l i s h Co. (N.Y.). 137. Townsley, C C , A.S. A t k i n s , and A.J. Davis. 1987. Suppression of p y r i t i c sulphur during f l o a t a t i o n t e s t s using the bacterium Thiobacillus ferrooxidans. B i o t e c h n o l . Bioeng. 30:1-8. 121 T r i b u t s c h , H. 1976. The o x i d a t i v e d i s i n t e g r a t i o n of s u l f i d e c r y s t a l s by T.ferrooxidans. Natur-wissenschaftern. 63:88. Tuovinen, O.H. 1978. I n h i b i t i o n of T.ferrooxidans by mineral f l o t a t i o n reagents. European J . a p p l i e d M i c r o b i o l . B i o t e c h n o l . 5:301-304. Tuovinen, O.H., and P.M. Sormunen. 1979. A note on the determination of ATP by the L u c i f e r i n - L u c i f e r a s e bioluminescence r e a c t i o n i n samples c o n t a i n i n g heavy metals. B u l l . Environ. Contam. T o x i c o l . 22:715-716. Tuovinen, O.H., P.R. Dugan, and A.A. D i S p i r i t o . 1983.-Sorp t i o n of Thiobacillus ferrooxidans t o p a r t i c u l a t e m a t e r i a l . B i o t e c h n o l . Bioeng. 25:1163-1165. T u t t l e , J.H., C.I. Randies, and P.R. Dugan. 1968. A c t i v i t y of microorganisms i n A c i d Mine Water: I . Influence of a c i d water on aerobic heterotrophs of a normal stream. J . B a c t e r i o l . 95:1495-1503. T u t t l e , J.H., and P.R. Dugan. 1976. I n h i b i t i o n of growth and i r o n and s u l f u r o x i d a t i o n i n T.ferrooxidans by simple organic compounds. Can. J . M i c r o b i o l . 22:719-730. T u t t l e , J.H., P.R. Dugan, and W.A. Apel. 1977. Leakage of c e l l u l a r m a t e r i a l from Thiobacillus ferrooxidans i n the presence of organic a c i d s . Appl. Environ. M i c r o b i o l . 33:459-469. Van Es, F.B., and L.A. Meyer-Reil. 1982. Biomass and metabolic a c t i v i t y of h e t e r o t r o p h i c marine b a c t e r i a , p. 111-170. In K.C. M a r s h a l l (ed.), Advances i n M i c r o b i a l Ecology. V o l . 6. Plenum Press, New York. Voger, K.G., and W.W. Umbreit. 1941. The n e c e s s i t y of d i r e c t contact i n s u l f u r o x i d a t i o n by Thiobacillus ferrooxidans. S o i l S c i . 51:331. Wakao, N., M. M i s h i n i , Y. S a l e u r a i , Y., and H. S h i o t a . 1983. B a c t e r i a l p y r i t e o x i d a t i o n I I . The e f f e c t of v a r i o u s organic substances on r e l e a s e of i r o n from p y r i t e by T.ferrooxidans. J . Gen. Appl. M i c r o b i o l . 29:177-185. Wakao, N., M. M i s h i n i , Y. Sakurai, and H. Shiota. 1984. B a c t e r i a l P y r i t e Oxidation I I I . Adsorption of T. ferrooxidans c e l l s on s o l i d surfaces and i t s e f f e c t on i r o n r e l e a s e from p y r i t e . J . Gen. Appl. M i c r o b i o l . 30:63-77. 122 149. Wassel, R.A., and A.L. M i l l s . 1983. Changes i n water and sediment b a c t e r i a l community s t r u c t u r e i n a lake r e c e i v i n g a c i d mine drainage. Microb. E c o l . 9:155-169. 150. Weerkamp, A.H., and B.C. McBride. 1980. C h a r a c t e r i z a t i o n of the adherence p r o p e r t i e s of Streptococcus salivarius. I n f e c t . Immun. 29:459-468. 151. Weertman, J . , and J.R. Weertman. 1964. Forces on a d i s l o c a t i o n . rn Elementary D i s l o c a t i o n Theory. Macmillan, N.Y. 152. Weiss, R.L. 1973. Attachment of b a c t e r i a to sulphur i n extreme environments. J.Gen. M i c r o b i o l . 77:501-507. 153. Wichlacz, P.L., and R.F. Unz. 1981. A c i d o p h i l i c , h e t e r o t r o p h i c b a c t e r i a of a c i d mine water. Appl. Environ. M i c r o b i o l . 41:1254-12 61. 154. Wichlacz, P.L., R.F. Unz, and T.A. Langworthy. 1986. Acidiphilium angustrum sp. nov., Acidiphilium f a c i l i s sp. nov. and Acidiphilium rubrum sp. nov.: A c i d i p h i l i c h e t e r o t r o p h i c b a c t e r i a i s o l a t e d from a c i d i c c o a l mine drainage. I n t . J . Syst. B a c t e r i a l . 36:197-201. 155. Wicken, A.J. 1985. B a c t e r i a l c e l l w a l l s and su r f a c e s , p. 45-70. In D.C. Savage and M. F l e t c h e r (ed.), B a c t e r i a l Adhesion: Mechanisms and P h y s i o l o g i c a l S i g n i f i c a n c e . Plenum Press. (N.Y.). 156. Yan, N.D. 1979. Phytoplankton community of an a c i d i f i e d , heavy metal-contaminated lake near Sudbury, Ontario: 1973-1977. Water A i r S o i l P o l l u t . 11:43-55. 157. Yeh, T.Y., J.R. Godshalk, G.J. Olson, and R.M. K e l l y . 1987. Use of e p i f l u o r e s c e n c e microscopy f o r c h a r a c t e r i z i n g the a c t i v i t y of Thiobacillus ferrooxidans on i r o n p y r i t e . B i o t e c h n o l . Bioeng. 30:138-146. 158. Zav a r z i n , G.A. 1972. A h e t e r o t r o p h i c s a t e l l i t e of Thiobacillus ferrooxidans. M i c r o b i o l o g y 41:323-324. 159. Zevenhuizen, L.P.T.M., J . D o l f i n g , E.J. Eshuis, and I . J . Scholten-Korselman. 1979. I n h i b i t o r y e f f e c t s of copper on b a c t e r i a r e l a t e d t o the f r e e i o n con c e n t r a t i o n . Microb. E c o l . 5:139-146. 123 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0098025/manifest

Comment

Related Items