Identification of bacteria recovered from acid mine environments by

reverse sample genome probing

Sophie A. Léveillé

Thesis subrni tted in partial fulfillmen t

of the requirement for the degree of

Master of Science in Biology (M.Sc.)

School of Graduate Studies

Laurentian University

Sudbury, Ontario

O Sophie Léveillé, December 1999 National Library Bi bliotheque nationale m*I of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON K 1A ON4 Ottawa ON KIA ON4 Canada Canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sel1 reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be p~tedor otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ACKNOWLEDGMENTS

I would like to take this opportunity to acknowledge my CO-supervisors,

Dr. L.G. Leduc and Dr. G.D. Ferroni, for their support, encouragement, and

confidence in me. I am very grateful to them for giving me the opportunity to

pursue my graduate studieç in their laboratory. 1 would like to thank cornmitte

member Dr. A.C. Ratiarson for a critical review of this thesis.

This project would have been impossible without the cooperation of Dr. G.

Voordouw who accepted to analyze the DNA samples. The success of this

project is also credited to his technicians, Anita Telang, who prepared the filters

and did the data analysis, and Yin Shen, who prepared the probes and did the

hybridizations. Thanks are extended to Dr. D.B.Johnson for generously

providing Lrptospirilliinifrrrooxida~zsCF12 and MK.

Many thanks to Danielle Monette for her help in the laboratory and for her

friendship, to Irene McAuley for her help in the laboratory, and to Willie

Desjardins for hs help with the pulverization of pyrite.

The assistance of Daniel Leduc in the laboratory and during the collection

of samples was greatly appreciated. Thank you for your support, advice,

patience, and friendship.

1 would also like to thank rny parents and family for their encouragement

and support throughout my studies.

Finally, 1 would like to acknowledge the National Science and Engineering

Council of Canada whose sdiolarship made it possible for me to pursue my s tudies. Aud mine drainage is an extreme environment. However, observations of the microbiota indicate that a variety of microorganisms can exist in such an environment. To elirninate acid mine drainage, and also to improve commercial bioleaching, it is imperative to identify the species of bacteria present in such environments. Reverse sample genome probing (RSGP) is a teclmique recently described for the identification of bacteria by DNA hybridization. The purpose of this investigation was (1) to isolate environmental strains of Thiobncilliis ferrooxidniis, T. ncidopliil iis, T. tliiooxidnns and Lep tospirill ilrn ferrooxidnns and (2) to applv RSGP to their identification in acid mine drainage samples.

Environmental strains of T. ferrooxidans and T. acidophilils were isolated and purified by repeated plating and single-colony isolation on iron salts and tetrathionate media, respectively. Different media were used to isolate

T. thiooxidnris, namely, thiosulfate, tetrathionate and sulfur media. For the isolation of Lrptospirilliim ferrooxidnris, iron sa1ts and pyrite media were inocula ted wi th environmen ta1 samples. However, L. ferrooxicintis was never recovered on solid media. Type cultures and environmental strains of

T.ferrooxidnns, T. ncidophilns, T. thiooricinns, and L. ferrooxidnns were used as standards and it was shown that RSGP was able to distinguish them.

Environmental samples were analyzed by RSGP after enrichment in different liquid media. Ferrous sulfate medium supported the growth of T. ferrooxidnns only, whereas al1 thiobacilli grew in sulfur medium, T. thiooxidnns being dominant. Growth in glucose followed by growth in tetra thionate resulted in the selection of T. ncidophilzls. The selected microorganisms were identifiable by

RSGP. DNA was also extracted directly (without enrichment) from cells recovered in acid mine drainage and sediments and analyzed by RSGP to describe the communities present in such environments. A better hybridization pattern was obtained when the environmental sample was prefiltered. TABLE OF CONTENTS

ACKNOWLEDGMENTS ABSTRACT

TABLE OF CONTENTS vi LIST OF TABLES viii LIST OF FIGURES INTRODUCTION Acid mine drainage Reactions of acid mine drainage Bioleaching Acidic mine environmen ts Tlziobncilliisfi.rrooxidms Thiobncillzis nci~lophilus Leptospirillirm firrooxidnns TIriobncilltis thiooxihrzs Isolation and identification of microorganisms Reverse sample genome probing Objectives MATERIALS AND biETHODS Type cultures Strain isolation Strain ailtiva tion Enrichment of environmental samples Environmen ta1 samples without enrichmen t Prepara tion of cells for DNA isolation DNA isolation Purification of DNA Speckropho tome tric analysis Preparation of con trois DNA shipment Preparation of master filters Preparation of DNA probes Hybridization of the probes to the filters Imaging and quantification of hybridization Qumti ta tive analysis of RSGP pattems RESULTS Strain isolation DNA purification Spectrophotometric analysis Quantitative analysis of RSGP patterns Cross-hybridization Analysis of controls by RSGP RÇGP analysis of environmental samples after enridunent Analysis of unidentified cultures by RSGP Analysis of rnicrobial cornmunites by RÇGP DISCUSSION DNA isolation protocol Isolation of T. thiooxidmzs Isolation of L. ferrooxidms RSGP analysis RSGP analysis of controls RSGP analysis of DNA samples after enridunent RSGP analysis of DNA samples fiom the environment The absence of L. Jerrooxi~in~isin environmental samples Conclusion REFERENCES APPENDICES Appendix 1: Culture media Appendix 2: Solutions Appendix 3: Spectrophotomehic raw data Appendix 4: Quantitative analysis

vii LIST OF TABLES

Table 1. Media used in the isolation of Thiobncillris thiooxidms from different water samples

Table 2. Media used in the isolation of Leptospirillzirn ferrooxidms

Table 3. DNA concentration For each standards included in mixture A

Table 4. DNA concentration of standards used in the preparation and probing of the filters

Table 5. DNA concentrations of samples used in the probing of the fil ters

Table 6. Gross colony characteristics of four strains capable of oxidizing thiosulfa te

Table 7. Growth of Leptospirillirrn frrrooxidnm MK and CF12 in different liquid and solid media

Table S. Incubation of a pyrite ciilture at different temperatures with or without shalung

Table 9. Absorbance at different wavelengths and purity of E. coli 11303 DNA recovered from 10 mL of culhne using different ex traction techniques

Table 10. Absorbance at different wavelengths and puritv of E. coli 11303 DNA reçovered hom 10 rnL of culture with0orwithout RNase treatment (done in duplica te)

Table 11. Self-hybridization constants of standards spotted on the mas ter filter LIST OF FIGURES

Figure 1. Protocol used to isolate environmental strains

Figure 2. Protocol used for the enridunent of environmental samples

Figure 3. Protocol used for RSGP analysis

Figure 4. Layou t of master fil ters

Figure 5.

Figure 6.

Figure 7.

Figure S. Cross-hybridization of T. t!iiooxidmzs ATCC 19377

Figure 9. Cross-hybridiza tion of T. frrrooxitinris ATCC 23270

Figure 10.

Figure 11.

Figure 12.

Figure 13. RÇGP of mixture A of standard genomes

Figure 14. RSGP of mixture B of standard genomes

Figure 15. RSGP of DNA extracted from an environmenta 1 sample enriched in TK medium

Figure 16. RSGP of DNA extracted from an environmenta1 sample enriched in Starkey's medium 1

Figure 17. RSCP of DNA extracted from an environmental sample enriched in tetrathionate medium

Figure 18. RSGP of DNA extracted from an environmental sample enriched in glucose medium which was then transferred to tetra thionate medium

Figure 19. RSGP of DNA extracted from an environmental sample enriched in glucose medium Figure 20. RSGP of DNA extracted from an environmental sample enriched in glucose medium (pH 2.3)

Figure 21. RSGP of DNA extracted from a thiosulfate-oxidizing strain Mlb 73

Figure 22. RÇGP of DNA extracted from a thiosulfate-oxidizing strain Mld 74

Figure 23. RSGP of DNA extracted from a thiosulfate-oxidizing strain M2aa 75

Figure 24. RSGP of DNA extracted from a tluosulfate-oxidizing strain M4b 76

Figure 25. RSGP of DNA extracted from a sulfur-oxidizing culture B (analysis 2) 77

Figure 26. RSGP of DNA extracted from a sulfur-oxidizing culture B (analysis 2)

Figure 27. RSGP of DNA extracted from an environmental water sample centrifuged to collect cells (analysis 1) 79

Figure 28. RSGP of DNA extracted from an environmental water sample cen trifuged to collect cells (analysis 2) 80

Figure 29. RSGP of DNA extracted from an environmental water sample prefiltered and centrifuged to collect cells 81

Figure 30. RSGP of DNA extracted from an environmental sediment sample INTRODUCTION

Acid mine drainage

blining of sulfide ores is very important econornically. However, it is the cause of serious environmentai problems. Every year, approximately 500,000,000 tons of tailings are deposited into engineered tailings sites covering roughly

15,000 hectares of Canadian lands (Davis et ri!. 1995). One problem confronting the mining industry is generation of acid drainage from sulfide-bearing mine wastes, including tailings and waste-rock piles. Tailings are a!lowed to drain after decommissioning, exposing sulfide minerals, such as pyrite, to oxygen and water. This exposure provokes chemical and microbial oxidation of these minerals with the production of sulfuric acid and soluble sulfates. As a result, the pH drops rapidly, killing aquatic life and rendering the contaminated stream unsuitable for human consumption and for recreational and industrial activities.

Another problem is the solubilization of toxic heavy metals, such as nickel and copper, which may be mobilized by bacterial leaching. The production of large amounts of sulfuric acid results in the mobilization of other available minerals in the surrounding rocks. Certain heavy metals, such as the major rock-forming element, aluminum, become soluble and contaminate effluents. Dissolved metals are toxic to aquatic life and may also be introduced into soi1 and ground water, if the ground beneath mine tailings is permeable.

The concentration of dissolved metals can reacfi elevated values in effluents with pH's lower than 3.0. Sedimentation is another environmental problem resulting from acid mine drainage. Sedimentation involves turbidity and deposition of iron (III) hydroxides (Fe(0H),). The iron (III) sa1ts can form fine suspended precipi ta tes, which can reduce the vision of consumer organisms, block gills and feeding mechanisms, and suppress light penetration reducing primary productivity

(Gray 1996). The iron precipitate will evenhlally settle out of suspension and encrust rocks and stones. As a result, the substrate in effluents is modified or destroyed and benthic life is eliminated. Consequentlv, a severe reduction in species diversi ty and population densi ty of the rnicrobio ta is observed.

Reactions of acid mine drainas

Pyrite, water, and oxygen represent the three basic components responsible for the production of acid mine waters. The reaction is generally sumrnarized as:

4 FeSz + 15 O2+ 14 H1O --> 4 Fe(OH), + 8 ÇO," + 16 H+ (1)

A pure ferric hydroxide phase is not the sole product of pvrite oxidation. In strongly acidic environments, a mixture of phases with variable stoichiometrv is produced, which includes jarosite (MFe,(S0,)2(0H),) and ferrihydrite

(Fe,O,SO, (OH),). Jarosite precipi ta tion is also an aad-producing reaction:

M++ 3 ~e"+ 2 HSO,- + 6 H,O -> MFe,(SO,), (OH), + 8 H+ (2) where M+is a monovalent cation. Moreover, other products, suc.as thiosulfate and sulfite, have been found to fom from pyrite oxidation. Elemental sulfur is another product that may result from pyrite oxidation, but the mechanism is not well understood.

Pyrite is the most abundant sulfide mineral in the Earth's crust. The main reactions in pynte oxidation are:

FeS, + 3.5 O? + H20--> FeSO, + Hz SO, (3)

2 FeSO, + 0.5 O2+ 5S04 --> Fe,(SO,), + H?O (4)

FeS,+ Fe,(SO,), ---> 3 FeSO, + 2 S (5)

2S+3o2+2H20--> 2 H2S0,(6)

Under abio tic conditions, pyrite oxida tion by atmospheric oxygen (reaction 3) occurs spontaneously and quite rapidly at a neutral pH, but its rate becomes relatively slow below pH 4.5 (Blowes et of. 1995). Therefore, in acidic environments, pyrite oxidation by ferric sulfate (reaction 5) becomes the dominant oxida tion mechanism. The rate of ferrous sulfate oxidation decreases with decreasing pH and reaction 4 becomes the rate-limiiing step in pyrite oxidation. However, the rate is completely different in the presence of bacteria.

As the pH drops, acidophilic bacteria (Thiobncill~rssp., Lrptuspirill~rnlfmrooxihns) take over by accelerating the rate of oxidation of ferrous sulfate and pyrite

(reactions 3-4). Ferric sulfate, the resul t of ferrous sulfate oxida tion, promo tes pyrite dissolution (reaction 5), consequently producing more ferrous sulfate and sulfur. Ferrous sulfate can then be rapidly oxidized again to yield ferric sulfate

(reaction 4), establishing a ferrous-femc cycle. Sulfur can be oxidized to produce sulfuric acid (reaction 6). The rate of microbial oxidation under low pH conditions is several hundred times higher than the spontaneous oxidation; a factor of 10' to IO6 times has been estimated (Tuovinen and Kelly 1973).

Bioleaching

Bioleaching is a result of rnicrobial metabolism, which converts insoluble metal sulfides to soluble metal sulfates. Bacteria of the genus Thiobncilll~s,as well as Lzptospirillwn frrrooxidms are recogmzed as being most active in bioleaching.

The activities of such microorganisms in dissolving minera1 deposits have important implications in the mining industry (Trudinger 1971). Ores containing metal sulfides such as zinc, lead, cobalt, nickel, bismuth, and antimony mav be leached through bacterial oxidation. Presently, commercial bioleaching is being used mainly for the recoverv of copper, uranium, and gold.

In the USA, approximately 10% of copper is obtained by bioleaching operations

(Yates et al. 1986).

Two mechanisms are possible for the bioleaching of metal-bearing ores.

The first mechanism involves bacterial attachrnent to the sulfide-minera1 surface followed by a direct oxidation of the mineral and the dissolution of the metal.

For example, chalcopyrite is oxidized to copper sulfate and ferrous sulfate according to the following reaction:

CuFeS, + 4 O2-> CuSO, + FeSO, (7)

in the indirect mechanism, bacteria generate a lixiviant which chernically oxidizes the sulfide mineral. In acid solution, tlus involves the ferrous-ferric cyde established by bacterial activity. Sulfuric acid and femc iron produced by bacterial oxidation of pyrite and other sulfide minerals can react with metal- bearing ore and solubilize the metal.

MS + 2 Fe3++ Hz + 2 0, -> M" + 2 Fe" + MSO, (8) where M is a bivalent metal

Bioleaching represents a useful alternative to conventional mining operations. For example, recoverv of metals from ores containing low mehl values or situated at great dep ths, by conventional mining techniques, can be a costly process. However, metal recovery through bioleaching can result in a profitable operation. Furthermorr, bioleaching of deep or low grade ores can be achieved iri sitii, thereb~eliminating the cost of transporting large amounts of ore to the surface. Bioleaching also represents an advantage over traditional extractive procedures for the processing of recalcitrant ores, ores that are difficult to treat. The recovery of gold in recalcitrant ores using conventional methods requires large quantities of energy compared to the use of bioleaching processes

(Lawrence 1990). Another advantage of rnicrobial leaching is that the outcome is less hazardous. For example, sulfurous ernissions do not occur and the process waste iç produced in either liquid or solid form and can therefore be contained.

Finally, the rates of sulfide minerais oxidation by bacteria are much faster than purel y chernical leaching mechanisms (Hackl1997).

Acidic mine environments

Acid mine drainage is often referred to as an extreme environment since its chernical nature does not allow colonization by a diversity of acid-intolerant microorganisrns. Although initially these environments were regarded as having lirnited species diversity, additional observations of the microbiota of acid mine drainage and mine waste environmen ts indicated that a sufficient varie ty of microorganisms and conditions could exist for diverse microbial interactions to occur (Norris and Kelly 1982). Such environments are now known to be populated by a range of acidophilic and acid-tolerant iron oxidizers, sulfur oxidizers, facul ta tive sulfur-oxidizing he terotrophs, and obliga te heterotrophs

(Harrison 1984; Norris 1990; Rawlings and Silver 1995; Schippers et al. 1995).

Most studies have focused on Thiobncillus spp. This genus of sulfur- oxidizing bacteria includes the obligate chemolithotrophic autotrophs,

T.jrrrooxidnns and T. thiooxidmzs, and the mixotrophs, T. ncidophillis and

T.criprinus, which are able to assimilate organic compounds as carbon sources while using inorganic compounds as electron donors. Leptospirillrm ferrooxidnns is another chemolithotrophic autotroph isolated from these extreme environmen ts. He terotrop hic bacteria commonly found in close relationship with the chernolithotrophs indude Acidiphili~lmspp. Acidobncterirr m cnpsrilnhlm is ano ther heterotroph inhabiting acidic environrnents. Finally, fungi, flagella tes, green algae, and yeasts have been also observed in such habitats.

Thiobncillt LS ferrooxidnns

Thiobncillzrs ferrooxidnm is probaoly the most important bacterium in bioleaching and acid mine drainage (Leduc and Ferroni 1994; Rawlings and

Silver 1995). This obligate chemolithoautotroph oxidizes and grows on sulfur, thiosulfate or tetrathionate and uses carbon dioxide primarily via the Calvin cycle as its major source of carbon for ce11 biosynthesis. In addition to oxidizing reduced sulfur compounds, this microorganism can oxidize ferrous iron, pmte, and numerous sulfide minerals during growth. This capacity allows it to play an important role in the bioleacl~ingprocess. T.ferroowidans can release metals from sulfidic ores by a direct or an indirect mechanism. 7'.frrrooxidnm is also the major microorganism in acid mine drainage since it increases the rate of acid genera tion by accelerating the oxida tion of ferrous iron.

T.ferrooxihns is usually described as a chernolithotrophic, acidophlic, and mesophilic Gram-negative bacterium. This rod-shaped microorganism occurs usually singly or in pairs, with a diarneter of 0.5 and a length of 1.0 Pm. It is motile by a single polar flagellum. Its growth temperature varies from 10 to 37'C with an optimum temperature in the range 30 to 35OC, depending of the strain.

However, psychrotrophic strains capable of growth on iron at temperatures varying from 2 to 37OC have been reported (Berthelot et nl. 1993; Ferroni et nl.

1986). Being a strict acidophile, T.ferroowirlnm prefers a pH of 2.0, but a pH range of 1.5 to 6.0 allows growth. T.fmooxidms is also a typical aerobic bacterium.

However, under certain conditions, it is capable of growing in anaerobic environments, oxidizing elemental sulfur to sulfuric acid using ferric iron as an electron accep tor.

Another property of T. ferrooxidnns is its resistance to many heavy metals like cobalt, lead, and alurninum. This resistance is very important when using rnicrobial activities in leadhg processes where the level of the solubilized metal can increase considerably. It has been shown that different strains show different sensitivities to certain metal ions such as nickel and copper (Leduc et al. 1997).

This variability is important since it allows the better adapted strains to survive and proliferate, which can be advantageous in bioleaching processes. Although

7'. ferrooxidnris shows resistance to most heavy metals, it is quite sensitive to uranium, silver, and mercurv ions (Robinson and Tuovinen 1984; Tuovinen et nl.

1971b).

Thiobncillzrs ~cihplzilzrs

Like T. frrrooxidnns, T. ncicioplzilirs is an aerobic, mesophilic, and acidophilic

Gram-negative bacterium. its growth temperature ranges from 25 to 37OC, but it prefers a temperature around 29°C. T. nciilophiiiis also needs an environment with a pH between 1.5 and 6.0, but its optimum growth occurs at a pH of 3.0.

These short rods occur singly, mainly in pairs, and rarely in chains. Motility is possible b y means of one flagellum or two subterminal flagella, but motility has no t been observed with al1 strains.

T. acicioophiltis is a facultative chemolithoauto troph. It can oxidize reduced sulfur compounds and elemental sulfur, and also grow heterotrophically on organic compounds such as glucose and sucrose. It can be transferred easily between glucose and sulfur media. Moreover, T. ncidophilris is well adapted to rnixo trophic grow th (Pronk rf al. l99Ob). Udike T. fwooxidnns, T. acidophil ils does not possess the capacity to oxidize ferrous iron (Arkesteyn and DeBont

1980; Guay and Silver 1975). Because of its metabolism, T.ncidophilils is often found as a contaminant in

T. frrrooxidczizs cultures. Surprisingly enough, T. nciriophiliis was carried for years

as a satellite of T. ferrooxidnns in iron salts medium without any organic

supplement. This behavior can be explained by the fact that 7'. acidophilils is

oligotrophic. In CO-culhirewith T. ferrooxidnns, it lives at the expense of organic

excretions, such as amino acids and alcohols, from T. ferrooxidms (Arkesteyn and

DeBont 1980). Such a cohabitation can improve metal extraction because it

prevents the accumulation of organic compounds which are often toxic to

T. frrroosirintis (Tuovinen et nl. 1971a; Tut tle and Dugan 1976).

L&os~irilli~nzferrooxicilrtts

Lep tospirill ilni ferroori~in~zsis a mesop hilic, acidophilic iron-oxidizing

bacterium that uses carbon dioxide as a carbon source. However, unlike

T.ferroosidrrris, it does not oxidize elemental çulfur or inorganic sulfur

compounds (Hallman et ni. 1993). Pvite can be used as energy substrate by the

bacterium. In fact, it seems to favor the growth of lep tospirilla (Battaglia et al.

1994). In a rnixed culture, leptospirilla overgrew T. frrroowidans during the

continuous bioleaching of pyrite (Battaglia et 01. 1994; Boon et nl. 1995; Goebel

and Stackebrandt 1991a; Helle and Onken 1988). L. ferrooxidnris is often

overlooked since it is less competitive in the usual isolation or enrichment media.

L. ferrooxidnns resembles T.ferrooxidms in several physiological properties,

but differs substantially in morphological characteristics. This spirillum is part of the farnily Spirillacene. Extensive polymorphism is typical for L. ferrooxicfnns (Pivovarova et al. 1981; Sand et nl. 1992). Such shapes as vibrios, helices with a different number of turns, pseudococci, and cocci are characteristic of L. ferrooxidnns, and are often the result of growth conditions. Battaglia et a!. (1994) noted vibrio-shaped cells under optimal conditions, spiral forms at sub-optimal temperature, and occasionally small coccoid cells, often aggregating. These changes in shape were also observed by Balashova et nl. (1974). According to

Sand rt al. (1992), cells have a diameter of 0.3 to 0.6 pm and a length of up to 3.5

Pm. Also, vibrioid cells of this iron oxidizer show rapid motility via a long single polar flagellum about 25 nm in diameter.

Since it was first described by Markosyan (1972), L. ferrooxihs has not been s tudied extensively. Physiological s tudies have shown variations in i ts optimum grow th temperature and pH. The optimum growth temperature for many strains appears to be between 20 and 30°C (Balashova et nl. 1974; Harrison

1984; Sand et al. 1992). However, a strain growing optimally at 3S°C (Harrison and Norris 1985) and several others having an optimal growth temperature between 37-40°C (Battaglia et al. 1994; Goebel and Stackebrandt 1994a) have been reported. As for pH, L. ferrooxidnrzs grows optimally at pH 1.5-2.0 (Hallrnan et 01.

1993; Rawlings and Silver 1995). However, it was suggested recently that

L. ferrooxidnns dorninated environments where conditions were generally above

40°C and pH was in the range 0.7 to 1.0 (Sduenk et RI. 1998). Overall, evidence shows that L. ferrooxidms has a higher tolerance for low pH values than

T.ferrooxidans (Battaglia et al. 1994; Sand et al. 1992) and that generation times of leptospirilla increase considerably at low temperatures (below 20°C) (Hallman et al. 1993).

Differences between the growth of L. ferrooxiiims and T. ferrooxidnns are not observed only in regard to pH and temperature values. In batch cultures, more extensive pyrite dissolution is noted for L. ferrooxidnns and the latter has a much greater tendency for attachment to pyrite (Norris et ni. 1988).

L. ferrooridnrzs shows a higher tolerance for high ferric iron concentrations than

T.ferrooxidnris (Norris et nl. 1988). Moreover, this microorganism also tolerates higher concentrations of uranium, molybdenum, and silver than T. ferrooxirinns.

However, it is more sensitive to copper and arsenate (Bosecker 1997; Harrison and Norris 1985; Said and Johnson 1988).

Tlziobncilltrs thiooxiiims

Thiobncillz~s thiooxidnris is an obligate acidophilic and chemolithoautotrophic bacterium that grows well on reduced sulfur compounds or elemental sulfur under acidic conditions producing sulfuric acid. Its major source of carbon cornes from carbon dioxide via the Calvin cycle. Cells oxidize sulfur to sulfate with sulfite as an intermediate: S0 --> SO," --> S0,'- (Suzuki

1965; Suzuki and Takeuchi 1992). Sulfuric acid can reduce the pH of a sulfur medium to values of 0.5 to 0.8.

Thiosulfate may also be a source of energy for this bacterium, which oxidizes both sulfur atoms to sulfate: S,O,'- + 2 O2 + H,O --> 2 S0,'- + 2 H'.

Also, growth on thiosulfate may produce, transiently, tetrathionate and sulfur (Chan and Isamu 1994). In the case of sulfur, thiosulfate is deaved into sulfur

and sulfite: S20,'- -4+ SO,". Sulfur is then oxidized to sulfite. However,

when the oxidation rate of sulfur is slower than the oxidation rate of thiosulfate,

sulfur is excreted outside of celis as elemental sulfur. As for the production of

tetrathionate, two molecules of thiosulfate are needed: 2 S20,'- --> S,0b2- + 2 e-.

Tetrathionate is oxidized further to sulfite, sulfur, and thiosulfate: S,O," + 2 e- --

> SO," + S + S203'- .

These short rods, 0.5 x 1.0 - 2.0 p,occur singly, in pairs or in short chains.

They are motile by means of a polar flagellum. The optimum temperature range

for growth is behveen 28 and 30°C with a growth range between 10-37°C. A pH

between 0.5 and 5.5 supports its growth, but its optimum pH is in the range 2.0-

3.0. T. tl~iooxi~ir~~zsis very similar rnorphologically and physiologically to T. ferrooxidnns, but it has been shown to be more acid-tolerant and more efficient at

oxidizing elemen ta1 sulfur (Hackl 1997).

T. thiooxidmis possesses a remarkable ability to oxidize elemental sulfur

rapidly. This ability makes it a major player in the cycling of sulfur in the

biosphere. It can also have an application in industry. This sulfur-oxidizing

bac terium can leach metals from various minerais. It cannot oxidize iron or

pyrite, but has been show to grow on sulfur from sulfudic ore in CO-culturewith

T.ferroosidnns (Sasaki et al. 1998) and Lrptospirill~~mferrooxidrtns (Sand et 01. 1992).

It can also play a role in desdfurization, the removal of pyritic sulfur from cm1

(Konishi et nl. 1995). Isolation and identification of rnicroorganisms

In order to eliminate acid mine drainage and to improve bioleaching, it is imperative to isolate and identify the species of autotrophic and heterotrophic bacteria present in such environments. Isolation of microorganisms from the environment requires a good knowledge of the target environment and its bacterial composition. The most common approach to isolate a microorganisrn involves the enrichment culture technique followed by purification using the streak plate method. A sample from the environment can be used to inoculate a

Liquid medium that selects for the target bacterium. Once growth occurs, the ctilture is transferred to a solid medium. Colonies are selected and transferred to a liquid medium. The culture is again plated, and single colonies are again selected to ensure purity. When growth on solid medium is problematic, serial dilutions can be employed by successively diluting a ce11 suspension in tubes of liquid medium and using the highest dilution showing growth as inoculum for the new set of dilutions.

A wide variety of identification methods are being applied to bacterial taxonomy. These methods include morphological and physioiogical characteristics, chernical analysis of bacterial components, such as proteins, polysaccharides and lipids, and the analysis of nucleic acids extracted from environmental isolates. A classic method involves the enrichment culture technique, where different culture media are inoculated with unidentified environmental isola tes and incubation conditions are chosen to favor the growth of those particular isolates. The absence or presence of growth is noted in each culture medium. It is then possible to classify the isolates according to their physiological characteris tics.

Many microorganisms found in mine tailings and effluents have similar morphologies, rnaking visual identification impossible. Characterization of these bacteria by cultivation methods is often difficult. Some grow extremely slowly in culture media and others grow poorly or not at al1 on solid media (Barns and

Nierzwich-Bauer 1997; Summers et al. 1986). T. frrrooxidmzs can be difficult to grow on iron salts solid media because it is sensitive to organic impurities found in agar or agarose and to sugars released from gelling agents due to acid hydrolysis (Tuovinen and Kelly 1973). In addition, methods that involve the cultivation of microbes are often time consuming and laborious. Hackl (1997) and Harrison (1982) acknowledged tha t acquisition of pure cultures of

T. ferrooxidmts is difficult and its maintenance is troublesome. Moreover, some of these fastidious bacteria may be ~mculhirableon any laboratory media. It has been estimated that 90-99" of microbial cells present in environmental samples are not culturable (Trevors and van Elsas 1995). These problems make isolation and identification of rnicroorganisms in mine effluents a formidable task (Yates and Holmes 1986a).

Reverse sam~lemnome ~robinp

Methods based on gene probes have been developed for the rapid and simple identification of microorganisms (Trevors 1985; Yates and Holmes 1986b).

DNA analysis can identify many of the bacteria present in the enviroiunent that conventional identification methods requiring culhiring may miss. Ya tes et RI.

(1986) were the first to use genetic probes to detect and identify acidophiles from biomining opera tions based on specific geno types and/or phenotypes. More recently, 165 rRNA oligonucleotide probes specific for sulfur- and/or iron- oxidizing microorga~smswere developed (Lane rt ni. 1992). Consequently, 16s rRNA probes were used to evaluate the microbial diversit~in commercial bioleaching si tes (Goebel and Stackebrand t 1994a), in natural sites (Goebel and

Stackebrandt 1994b) and in biooxidatioii tanks (Rawlings 1995). Bacterial populations have also been characterized by analyzing the spacer regions between the 16 and 23s rRNA gene (Pizarro et ni. 1996) or by using small-subunit rRNA sequences (Schrenk et ni. 19%).

Reverse sample genome probing (RSGP) is a DNA hybridization technique that has recently been used for the identification of bacteria in environmental samples (Voordouw et ni. 1991). It involves dena tured chromosomal DNA of a given bacterium which rnay be used to confirm its presence in labeled total genomic sample DNA. This is possible since the genomes of different bacteria may have little overall sequence homology and therefore, whole genome probes can, in many cases, distinguish species even within the same genus (Shen et al. 1998; Voordouw et nl. 1991). The term reverse sample genome probing was adopted to describe this technique because it represents the reverse of the usual practice which labels specific bacteria DNA to analyze DNA recovered from an environmental sample. In RSGP analysis, chromosomal DNA isolated from bacteria obtained from the target environment or from type culture collections is spotted on a membrane, referred to as master filter. Next, genomes of these bacteria are hybridized with each other. Ideally, little or no cross-hybridization should be observed within the genomes spotted on the master filter. When this occurs, the isolates are referred to as bacterial standards. The self-hybridization constants can then be determined wi th the resul ts of cross-hybridiza tion. Finally, to ta1

DNA extracted from an environmental sample can be labeled and used to probe the master filter. The radioactivity in each spot can be quantified to identify which of the bacterial genomes spotted on the filter are most common in the sample.

The RSGP method has many advantages. The development of a master fil ter spot ted wi th sui table standards results in the identification of mu1 tiple miaoorganisms in a sample in a single step. Another advantage is the need for only a small amount of denatured chromosomal DNA (-10 ng) per filter.

Therefore, 1 mg of chromosomal DNA from a standard allows the production of

10' filters. Also, the membranes used to make master filters have a high capacity to bind DNA. This capacity perrnits the minute amount of DNA to be confined to a very small area. In principle, 10 x 10 cm membranes could be spotted with

IO4 standards. Such master filters could give much information on microbial diversi ty of environmental samples. Obiec tives

The objectives of this research were (1) to isolate environmental strains of

Thiobncillits ferrooxidms, T. ncidophilils, T. thiooxidnns, and Lrptospirillrirn ferrooxidrins and (2) to apply RSGP to the identification of bacteria (T. ferrooxidnns,

T. acidophihs, T. thiooxidnizs, and L. ferrooxidans) recovered from acid mine effluents. MATERIALS AND METHODS

Twe culhires

A type culture of each species, except Leptospirillrim ferrooxidnris, was

ob tained from the American Type Culture Collection (ATCC). Thio bncilliis ferrooriiimis ATCC 23270 was maintained in iron salts (TK) liquid medium

(Appendix 1, Table 1) and T. ncidopliiltls ATCC 27807 was maintained in

tetrathionate liquid medium (Appendix 1, Table 2). As for T.thiooxidnns ATCC

19377, Starkey's Medium 1 (Appendix 1, Table 3) was used for its maintenance.

Two strains of L. férrooxidnris (MK and CF12) were gifts from Dr. D. B. Johnson,

University of Wales (Bangor), U.K., and these were maintained in TK liquid

medium.

Strain isolation

A sample of water (lO.-I°C, pH 3.4 was collected from an acid mine

effluent in Copper Cliff, Ontario that emerged from INCO's Copper Cliff Tailings

Area and entered Copper Cliff Creek. The sample was used to isolate

environmental strains of T.frrrooxidnizs, T. neidophilus, T. thiooxidnns, and

L. ferrooxidnns by enridunent in different culture media.

Figure 1 summarizes the method used for the isolation of T. ~ciduphilzis.A

volume of 100 mL of environmental water was filtered through a Nalgene

150-mL analytical filter unit. The filter membrane was then transferred to 100

mL of te tra thionate medium to recover T. czcidophihs. The 250-mL Erlenrneyer environmenta1 fil ter unit culture medium water sample

agarose plates repea ted

culture medium

Figure 1. Protocol used to isolate environmental strains flask was incubated for up to one week on a gyratory shaker (200 rpm) at 22OC.

After serial dilution in 10 mL of tetrathionate liquid medium and addition of

50 PL of sterile Tween 80, a 0.-mL volume of growing culture was plated ont0 tetrathiona te solid medium (Appendix 1, Table 2). Colonies tha t were smooth, white with a yellow center and 1 to 2 mm in diameter, after a three week incubation period at E°C, were selected and inoculated into 100 mL of tetra thiona te liquid medium. The cultures were again pla ted, and single colonies were selected to ensure purity. Identification of the isolates was achieved by microscopic analvsis and presence of growth in different liquid media. Seven liquid media were prepared by replacing tetrathionate in the tetrathionate liquid medium with copper sulfide, citric acid, glucose, lactose, mannitol, sucrose, and thiosulfate. In addition, the isolates were inoculated into TK liquid medium to determine if they had the capacity to oxidize ferrous iron.

A sirnilar protocol was followed to acquire T.frrrooxidms in pure cultures.

The membrane filter was introduced in TK liquid medium and the culture was plated ont0 iron salts purified (ISP) agarose medium (Appendix 1, Table 4).

Small rust-colored colonies were selected and cultivated in liquid medium.

Again, purification of cultures was achieved by repea ted plating and single- colony isolation.

Medium S (Appendix 1, Table S), tetrathionate medium, Waksman medium (Appendix 1, Table 6), and 77ziobncilliis Medium B (Appendix 1, Table 7) were inoculated with different environmental water samples to recover

T. thiooxidnns. The sample was filtered and the membrane filter was introduced in the different media. These cultures were then transferred to solid media such as Medium S (Appendix 1, Table 5) and tetrathionate, both solidified with 1% agarose (w/v), to attempt to isolate colonies typical of T. thiooxidnm. Table 1 summarizes the media used for T. thiooxicilrns isolation from different water samples. Also, the method of highest positive dilution was used for the isolation of T. thiooxidms in pure cultures as follows. Serial dilutions were made in 10-fold steps up to IO-'' using Çtarkev's Medium 1 and the tubes were incubated at 22OC.

The inoculum for the first set of tubes consisted of an environmental sample enriched in Starkev's Medium 1. The highest positive dilution was used to inoculate another set of tubes. Transfers were repeated five times.

For the growth of Lt.ptospirillirrn firrooxidnns, TK and pyrite (Appendix 1,

Table 8) liquid media were inoculated with water or sediments from mine tailings effluents or a colony on ISP. After growth was apparent, these cultures were then transferred to the following solid media: FeTSB (Appendix 1, Table 9),

ISP 0.3% FeSO,, ISP 1.0% FeSO,, and ISP 3.3% FeSO,. Table 2 summarizes the different media used to recover L. ferrooxihns. For example, a water sample was used to inoculate TK medium and the developping culture was plated onto ISP

0.3% FeSO,. Pure cultures were also established by continued serial dilution and subcultured in pyrite medium (pH 1.8). Serial dilutions in pyrite medium were made in 10-fold steps up to and repeated at least twice using the highest positive dilution step. The first set of tubes was inoculated with the pyrite medium described in Table 2 and incubated at Z°C. Table 1. Media used in the isolation of Thiobacillzis thiooxidms from different water samples

water sample medium 10.4"C, pH 3.40 Tetra thionate 10.4"C, pH 3.40 Tetrathionate, Medium S 10.4"C, pH 3.40 Waksman, Medium S 3.3"C, pH 2.79 TFziobacilllis Medium B, Medium S 15.1°C, pH 3.96 Medium S

The Thiobncillus Medium B culture was transferred to Waksman before being used to inocula te Medium S. Table 2. Media used in the isolation of Lep tospirill ir rn ferrooxidnns

inociilum liquid medium solid medium water (lOA°C, pH 3.40) ISP 0.3% FeSOa sediments (10.4"C, pH 3.40) ISP 0.3% FeSOa

sedimen ts (28.1°C, pH 2.85) pyrite ISP 0.3% FeS04 colony on ISP TK FeTSB water (1.7"C, pH 2.82) [SI? 1.0% FeSOa water (1.7"C, pH 2.82) ISP 3.3% FeSOa Since isolation of L. ferrooxidizns from the environment proved to be problematic, two type cultures of L. ferrooxihns, MK and CF12, were grown on a variety of media. TK liquid medium, pH 1.8, 2.1 and 3.0, Thiobncillzis medium

(Appendix 1, Table IO), and 1% pyrite medium were inoculated with 1% (v/v) liquid sample of MK and CF12 Also, solid media, such as Thiobncillns medium,

FeTSB, ISP 0.3'' FeSO,, ISP 1.O0k FeSO,, ISP 3.3% FeSO,, TK 3.300 FeSO,, and TK

5% FeSO, (Appendix 1, Table 11) were tested for colony formation.

Strain cultiva tion

Cultures of T. ferrooxidntzs and L. ferrooxidnns to be used for DNA isolation were grown by inoculating (1%;v/v) TK liquid medium, pH 1.8. Such a low pH reduced the reaction between Fe'+ and phosphate, which forms a precipitate, and the formation of insoluble ferric hydroxysulfate from Fe3' generated during growth (Harrison 1984). Cultivation of T. ncidophilus was perforrned in the same mmer in 0.5% (w/v) glucose medium, pH 3.2. This glucose medium consisted of the tetrathionate medium with glucose replacing tetrathionate. Growth of the

T. thiooxihm type culture was done in tetrathionate and putative T. thiooxidnns environmental cultures were grown in Medium S. Enrichment cultures were incubated for 1 to 4 weeks on a gyratory shaker (200 rpm) at 22T until a color change or turbidity occurred in the medium. Enrichment of environmental sam~les

In addition to the isolates, water samples (23.0°C, pH 2.6) from INCO mine tailings effluents were collected and filtered through a Nalgene 150-mL analytical filter unit. The membranes were used to inoculate 1 L of four different liquid media. A TK liquid medium (pH 1.8) favored the growth of T.fenooxidnns and L. ferrooxidms. Çtarkey's Medium 1 and tetrathionate medium were used for the growth of T. tlzioowidn~ls,T. ferroosidczris, and T. ncidophilzrs. To ensure the selection of T. midophillis, a second medium containing 0.5% glucose instead of tetrathionate (pH 3.2) was used. In the first case, a tetrathionate medium was inocula ted wi th an environmental sample and when gowth occurred, an aliquo t was translerred to a glucose medium. In the second case, an environmental sample was introduced into a glucose medium, which was then transferred to tetrathionate medium. Also, a glucose medium (pH 2.3) was inoculated with 1%

(v/v) of Starkey's Medium 1 previously inoculated with an environmental sample. Figure 2 depicts this protocol. Incubation of al1 inoculated media, except Starkey's Medium 1, was done on a gyratory shaker (200 rpm) at 22OC for up to two weeks. Starkey's Medium 1 was incubated without shaking and once growth occurred, sulfur was removed by filtration through a Whatman No. 1 fil ter paper under suction.

Environmenta1 samples wi thou t enrichment

Environmental sarnples (l°C, pH 3.0) were obtained from the same location described previously. Water samples (1 liter each) were collected in 20 environmental 1 wa ter sarn ple j filter unit

TK medium Starkey's Medium 1 tetrathionate glucose

glucose (pH 2.3) glucose tetrathiona te

Figure 2. Protoc01 used for the e~~hmentof environmental samples sterile plastic bottles, closed with a screw cap, and transported to the laboratory at ambient temperahire. Sample processing started within 24 h of sample collection. Liquid samples were centrifuged in 250-mL Nalgene bottles at

25,900 x g for 10 min at 4'C. The supernatant was discarded and the pellets were left in the bottles until 20 L were centrifuged. The resulting pellets were combined in a microcentrifuge tube, which was stored at -70°C until DNA extraction was performed.

Additional water sarnples (l°C, pH 3.0) were collected at the same site in a

50-L Nalgene carboy and processed within a few hours of sampling. The bacterial fraction was isolated from water by using a modification of the technique described by Fuhrman et ni. (1988). The environmental water was prefiltered through non-sterile Whatman No. 1 filter paper with a diameter of 90 mm to remove most eucaryotic rnicroorganisms. Cells from 20 L were collected by centrifugation as described previousl~. Cells from another 20 L were collected by filtration through 0.45-v-pore-size nitrocellulose membrane filters

(Micron Separations). A filter with a diameter of 47 mm was used to filter 5 L of water. The four filters were then stored in sterile Petri dishes at -20°C until extraction. For extraction of DNA, the frozen filters were cut with a clean razor blade into 2 mm x 1 cm strips and added to 2 mL of TE buffer (10 mM Tris HCl,

0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0) in 50-mL oak ridge fluorina ted e thylene propylene (FEP) centrifuge tubes (Nalgene). DNA isolation and purification followed the protocol described later except the volumes were adjusted accordingly. Finally, sediments found at the bottom of acid mine drainage effluents were collected in a sterile 1-L Nalgene plastic bottle. Isolation of the bacterial fraction was started after 24 h of sediment collection using a modification of the procedure described by Torsvik et nl. (1995). The water was drained from the sediments and 45 g of sediment were transferred to twelve 250-mL Nalgene centrifuge bottles. A 150-mL volume of sterile water (pH 3.0) was added to each bottle and the bottles were shaken on a platform shaker at 250 rpm for 1 h. They were then centrifuged at 1,000 x g for 15 min at 4OC. The supematants were pooled and stored at AOC. Again, 150 mL of sterile water were added to the sediment pellets and homogenized for 10 min. The homogenate was centrifuged again at 1,000 x g for 15 min at a°C and the supematants were combined with the earlier pooled supernatants. The procedure was repeated a third time. The combined supernatants were centrifuged at 35,900 x g for 10 min at PC. The supernatant was discarded and the pellets were left in the bottles until the total volume had been centrifuged. The final pellets were resuspended in 1 mL of TE buffer and stored at -70°C until DNA extraction.

Pre~arationof cells for DNA isolation

Once the cultures were grown, cells were collected by centrifugation at

25,900 x g for 10 min at 4OC. Cells were washed twice by resuspending the pellet in 10 mL of culture medium without the electron donor or acidified water

(adjusted to the appropriate pH with H,SO,) and centrifuging again. Cells were then resuspended in 1 mL of TE buffer and transferred to a 1.5-mL rnicrocentrifuge tube. Samples from iron salts cultures were left unrnoved for approximately 10 min to allow jarosite to settle at the bottom of the tube. The supernatant was then collected and transferred to another microcentrifuge tube.

The optical density obtained at 650 nm was adjusted between 8 and 12 for each isolate and sample. A volume of 100 mL of ce11 supsension was transferred to

900 mL of TE buffer and the optical densitv kvas obtained, taking into account the dilution. If the optical density was not between 8 and 12, the ce11 suspension was either diluted or concentrated accordingly and the optical density was verified.

When the appropriate optical density was obtained, cells were immersed in liquid nitrogen and kept at -70°C until DNA isolation.

DNA isolation

DNA was extracted from cells by the Marmur (1961) method modified as described here. DNA isolation started by centrifuging the tubes containing washed cells at 8,944 x g for 10 min at PC. The supernatant was decanted and the pellet was resuspended in 0.5 mL of 0.15 M NaCl, 0.1 M EDTA, pH 8.0. Cells were pelleted again by spiming at maximum speed (15,115 x g) for 5 min at 4'C.

Again, the pellet was resuspended in 0.25 mL of NaCl/EDTA solution. A 10-PL volurne of a 10 mg/mL lysozyme solution and 20 PL of a 25% sodium dodecyl sulfate (SDS) solution were added. Cells were incnibated at 60°C for 15 min, then transferred to -70°C for 15 min, then back to 60°C for another 15 min. The freeze-thaw cycles were continued until the supernatant began to look dear, which was observed after about 3 cydes. The addition of 60 pL of 5 M NaC10, and 300 pL of chloroform-isoamyl alcohol (24:l) followed the last incubation at

60°C. The mixture was mixed gently on a rotating wheel for 1 h and then centrifuged at maximum speed (15,115 x g) for 2 min. The upper layer was carefully transferred to a new tube, avoiding the white interface, and two volumes (about 800 PL) of 95% ethanol were added to the transferred top layer, precipitating DNA as a white clump. The microcentrifuge tube was stored at

-20°C overnight.

The nucleic acids were collected by centrifuging at maximum speed

(15,115 x g) for 15 min at -I°C. The pellet was resuspended in 400 pL of TE buffer, in which 10 PL of 10 mg/mL DNase-free RNase (Appendix 2) was added. The tube was then incubated for 30 min at ZaC.

Purification of DNA

To purify the DNA samples, four different techniques were tested on cells recovered from 10 mL of an Escherichin coli ATCC 11303 culture. In the first technique, DNA was extracted twice with 150 PL of 24:l chloroform-isoamyl alcohol. As for the second protocol, extraction was done once with 150 pL of TE- saturated phenol and then once with 150 pL of chloroform. The third technique consisted of extracting DNA once with 150 PL of TE-saturated phenol and then twice with 400 pL of diethyl ether. Finally, DNA was extracted twice with 400 pL of TE-saturated phenol-chloroform-isoamyl alcohol followed by a third extraction with 400 pL of diloroform-isoamyl alcohol. For each extraction, the tube was rnixed by inversion for 5 to 10 min and centrihged at 15,115 x g for 10 min at 4T. The upper phase was rernoved and transferred to a new tube in which the extractant was added. Extraction with chloroform-isoamyl alcohol was repeated up to three times, depending on the amount of protein seen at the interface. After the last extraction, DNA was precipitated with 95% ethanol by adding two volumes (800 ILL) of ethanol and stored at -20°C. The extraction technique giving the beçt results for DNA purity was chosen for further DNA p~uification.

The effect of RNase treatment was tested in duplicate on E. coli cells recovered kom 10 mL of a nutrient broth culture. Cells were lysed according to the proced~iredescribed earlier. A 10-/AL volume of 10 mg/mL DNase-free mase was added to two tubes which were then incubated for 30 min at room temperature. Afterwards, RNase-treated and non-treated DNA samples were purified with 24:l chloroform-isoamyl alcohol. DNA samples were precipitated with two volumes of ethanol and stored at -20°C. Spectrophotometric analyses were then perforrned and the technique with the best results for purity was chosen for further DNA ex tractions.

Soectro~ho tome tric anal ysis

After an ovemight incubation at -20°C, DNA was spun at 15,115 x g for

15 min at 4Tand resuspended in 500 PL of TE buffer. The concentration and purity of DNA was deterrnined by measuring absorbance at wavelengths of

260 nm and 280 nm using a Beckman W/Vis spectrophotometer (mode1 DU-65).

The reading at 260 nm allowed calculation of the concentration of DNA in mg/mL by dividing the value obtained by 20. This is possible since an optical density (OD) of 1 corresponds to approxirnately 50 pg/mL for double-stranded

DNA. The ratio between the readings at 260 nm and 280 nm (OD,/OD,) provided an estimate of the purity of DNA. For most DNA samples, the background correction provided by the reading at 320 nrn was included in the ratio. Pure preparations of double-stranded DNA had an OD,,/OD,o of approxima tely 1.8. After spectrop hotometric analysis, DNA was precipita ted in

95% ethanol and stored at -20°C until further steps were performed.

Pre~aration of controls

To ensure that the isolates would be identifiable in a sample by RSGP analysis, two mixtures including DNA from the eight isolates were prepared as controls. One control consisted of DNA obtained from the eight isolates which were mixed together before DNA extraction (mixture B). Cells of each isolate were collected bv centrifugation in separate tubes and suspended in TE buffer, such that each culture had comparable concentrations. A 0.125-mL volume of each culture was then transferred to a microcentrifuge tube and 0.1 mL of TE buffer was added. The optical density obtained at 650 nrn was adjusted to between 8 and 12. Then, DNA was extracted and purified as described earlier.

The other mixture consisted of extracting DNA from the eight isolates individually and then combining that DNA in a single tube (mixture A). The concentration of DNA recovered from each isolate and the volume transferred to the tube are included in Table 3. Approximately 15 pg of DNA from each isolate Table 3. DNA concentration for each standard included in mixture A

DNA concentration DNA volume DNA standard (~g/mL) (PL) amoun t (pg) T.ncidophilils ATCC 27807 70.03 214 14.98 T. ncidophilz~s64 45.95 326 14.98 T.acidophilils 46 90.52 166 15.03 T. thiooxi~imsATCC 19377 23.83 500 11.92 T.ferrooxi~ims ATCC 23270 56.94 263 14.98 T. frrrooxidtzns FI 32.60 100 3.26 T. ferrooxidnris F2 26.62 500 13.31 L. ferrooxihns CF12 53.04 283 15.O1

T. nciciophiliis 64, T. ncidophilils 46, T. frrrooxidnns FI, and T. frrrooxidnns F2 originated kom acid mine environments. were added to that single microcentrifuge hlbe (Table 3). The concentration and purity of both mixtures were detemined by spectrophotometric analysis.

DNA shi~ment

Precipitated DNA samples were centrifuged at 15,115 x g for 15 min at d°C and the supernatant was discarded. Microcentrifuge tubes were left opened at room temperature until the ethanol evaporated. DNA samples were then shipped to the University of Calgary as dried pellets for RSGP analysis. The remainder of the protocol was done bv technicians at the University of Calgary under the supervision of Dr. C. Voordouw. Figure 3 summarized the RSGP procedure. The resul ts were later forwarded to us.

Premra tion of rnaster fil ters

Grids of 6 x 2 squares were drawn in pend on 20,l cm x 1 cm Hybond-N hybridization membrane fil ters (Amersham). DNA samples from type cultures and environmental strains were used as standards. Dried DNA pellets were resuspended in TE buffer using volumes listed in Table 4, which also includes their concentration. DNA aliquots of standards were further diluted in TE buffer to a total volume of 60 pL (Table 4 - spotted conc). 1/5 dilutions of this 60 pL were also used to make probes and their concentrations are recorded in Table 4.

Bacteriophage h DNA (Pharmacia, 500 ng/pL) was diluted in TE buffer to concentrations of 0.5,5,10 and 20 ng/pL. A env ironmental sample * 1selective enrichmen t environmental s trains

env ironmental samples

enrichmen t G H b fi ÂlO 120 1 extraction 4 --- 1 mas ter fil ter

add intemal standard la bel self-hybridization constants hybriQze with master filter

analy ze hy bridization pa t tem 1wi th phosphoimager

0.5 quantitative R!5GP

ABCDE FGH standard

Figure 3. Protocol used for RSGP analysis Table 4. DNA concentrations of standards used in the preparation and probing of the filters

standard TE DNA DNA DNA volume concentration spo tted conc probe conc (PL) (ng/pL) (ng/W (ng/pL)

------T.ncidophilr~s ATCC 27807 T.ncidophiliis 64 T. ncidopliilzls 46 T. thiooxidms ATCC 19377 T.frrrooxihizs ATCC 23270 T.ferrooxihns FI T.firrooxihns F2 L. ferrooxidnizs CF12

T. ncidophiliis 64, T. ncidopliiliis 46, T. frrrooxidntzs FI, and T. /errooxidms F2 originated from acid mine environments. The DNA standards and the h DNA were placed in a boiling water bath.

After 2 min, they were removed directly to an ice/water bath. Once they had cooled, they were centrifuged briefly to collect the sample at the bottom of the tube and placed again on ice. A volume of 2 PL of each DNA was spotted on the

Hybond grids using a Hamilton shown dispensing pipette. The spotting was done according to the template displayeci in Fig. 4 such that al1 20 blots were identical. Table 4 shows the concentrations used to spot each isolate (spotted conc).

The dot blots were left to dry at 22OC for a period of 3 h. Then, they were

UV irradiated at 312 nm for 3 min to link the DNA to the filters. Following the cross-linking, the filters were washed in 1X SSC buffer (0.15 M NaCI, 0.015 M

Na,C,H,OJ for 10 min. Again, the dot blots were left to dry overnight at room tempera h~re.

Pre~arationof DNA probes

DNA pellets extracted Crom environmental samples with or without enrichment were resuspended in TE buffer according to Table 5, which also lists their concentrations. The DNA samples were then diluted to the concentrations listed in Table 5 (probe conc). DNA from environrnental samples and standards were used as probes. The following mixture was prepared for al1 probes. A volume of 10 PL of genomic DNA was added to 5 PL of H20 and 5 PL of

0.5 ng/@ h DNA. The tubes were placed in a boiling water bath for a period of

2 min. They were then removed to an ice/water bath until they had cooled. A L

A B C D E F

G H no DNA A5 hl0 A20

Figure 4. Lavout of mas ter filters

Legend

A T. ncidophilus ATCC 27807 B T. ncidopliihis 64 C T. nciciophilirs 46 D T. thiooxihns ATCC 19377

T. rrciciophil irs 64, T. izcidopliil ils 46, T. frrrooxi~inrzs FI, and T. ferrooxidms F2 origina ted from acid mine environmen ts. Table 5. DNA concentrations of samples used in the probing of the filters

DNA samples TE volume DNA DNA probe (CIL) concentration conc (ng/~iL) (ng/m mixture B mixture A e~chmentin iron salts (1.8) enrichment in siilfur enrichment in tetrathiona te enrichment in glucose (3.2) then in tetrathiona te enrichment in glucose (3.2) enrichment in sulfur then in glucose (2.3) thiosulfa te-oxidizing isola te thiosulfa te-oxidizing isolate thiosulfa te-oxidizing isola te thiosulfa te-oxidizing isola te sulfur-oxidizing isola te centrifuged wa ter sedimen ts prefil tered and cenhifuged wa ter

Values within brackets represent the pH of the medium. volume of 6 pL of primer extension (PE) mix (Appendix 2) was added to each tube, followed by 2 yL of Klenow DNA Polymerase (1 U/lI,, Pharmacia) and 2 pL of [c(-~~P]~CTP(>3,000 Ci/mmol, 10 mCi/mL, ICN). Each tube was briefly centrifuged to collect the sample at the bottom of the tubes. Then, for at least 2 h

(usually 4, the tubes were Left at Z°C, behind shields to radiolabel. Probes were prepared in two sets on two different days.

Hvbridization of the robes to the filters

Filters were prehybridized wi th 2.5 mL of prehybridization solution

(Appendix 2) in individual Ziplock plastic bags, trimrned to fit the filter and sealed with a bag sealer. Prehybridization was done at 6S°C with gentle shaking for 4 to 5 h. Then, the bags were cut open but the prehybridization solution was not removed from the bags. Before adding the probes to the prehybridization solution in the bags, they were first boiled for 2 min, removed directly to an ice/water bath and then centrifuged briefly. Once the probes were added, the bags were resealed and incubated ovemight at 6S°C for hybridization.

Sixteen to eighteen hours after adding the probes, the bags were cut open and the filters were removed and placed in plastic boxes containing wash solutions. Filters from each set were al1 washed together in one box. The first wash solution was 100 mL of 1X SSC. Filters were incubated in this solution for 5 min at room temperature while gently shaking. The first solution was then poured off and 100 mL of 1X SSC solution was added. The filters were washed again for 5 min at room temperature. Following the second wash, the solution was rernoved and replaced with 1X SSC, O.ZO/O SDS (lx SSC + 0.2% weight/volume SDS). Filters were gently shaken h this solution at 68OC and the solution was poured off after 1 h of incubation. Filters were then hansferred to 3

MM paper (Whatman) and left to dry for 15 to 20 min. When the filters were completely dry, they were wrapped in Satan wrap (Dow) for imagmg.

Imaeing and auantifica tion of hvbridization

Radioactive filters still wrapped in Saran wrap were exposed to a Fuji BAS

1000 imaging plate. The exposure took place for 2 h. The Fuji BAS 1GûO phosphoimager %vasthen used to scan the plate and transfer the images on a

Power Macintosh cornputer. The images were acquired and hybridization intensities for al1 of the dots were deterrnined using MacBas 2.2 software.

For every filter, each position on the filter was circled whether or not hybridization occurred and its photostimulable luminescence value was obtained. The data for each filter was then processed with Microsoft Excel5 for further anal ysis.

Quanti ta tive analvsis of RSGP ~a t tems

The net hybridization intensities for al1 the radioactive spots (1, and II) were determined in mits of photostimulable luminescence (PSL) by subhacting a local background. The fractions f, of al1 genomes were calculated from the hybridization data according to equation 9 (Voordouw et al. 1993):

f, = (kJk,) x (IJc,) x (I,/cJL x f, (9) where k, and k, are hybridization constants; f, and fi are the weight fractions of standard x and bacteriophage )c DNA in the probe that hybridized with immobilized standards x and A; c, and c, are the weights of denatured DNA x and h spotted on the filter; and 1, and IL are the net hybridization intensities.

The ratio (k,/ k,) was analyzed with equation 10:

!k,/k,) = (kj.,l,., x f, x c,) / 1, (10) k,,,, is obtained from the average of the h data (11):

k*,,, = 1, / (fi x c,) (11) ft and f, are the fraction of genornic DNA in the probe (e.g. 100 ng of DNA for standard x with 2.5 ng of h DNA combined in the labeling reaction results in f, =

0.976 and f, = 0.024). RESULTS

Strain isolation

The water sarnples collected from aad mine effluents showed a high level of acidity (pH 3.4). Following inoculation into TK medium, turbidity and colour change were observed after one week of incubation. Two strains, F1 and F2, were isolated from those effluents and purified by repeated plating and single- colonv isolation. The capacity of F1 and F2 to oxidize ferrous iron and the formation of rust-coloured colonies on solid medium are typical of

T.ferrooxillnns.

Inoculation of tetrathionate medium with a water sample resulted in growth after approximately a week of incubation. Growth was noted by the presence of turbidity. Several strains were isola ted and purified b y repeated subculturing of single colonies from solid to liquid media. The isolates were capable of oxidizing five of the eight substrates, namely citric acid, glucose, mannitol, sucrose, and thiosulfate. Growth was not observed in copper sulfide, lactose, and TK media. These physiological characteristics confirmed that the isolates were T. acidopliiliis. Isolates 46 and 64 were chosen for RSGP analysis.

For T. thiooxihns, growth in liquid medium usually occurred within one week and growth on solid medium occurred within two weeks. Growth in liquid medium was indicated by turbidity. Most strains were lost after a transfer from solid to liquid medium. However, four strains (Mlb, Mld, Waa, and M4b) were isolated using Medium S liquid and solid media via colony formation. Table 6 shows the colony characteristics of the thiosulfate-oxidizing strains.

Serial dilutions were also done in sulfur medium to isolate T. thiooxidiins.

Growth was deterrnined by turbidity and microscopie analysis and the highest positive dilution step was used to inoculate another set of tubes. After repeating the transfer five times, culture B was obtained. The thiosulfate-oxidizing strains and the sulfur-oxidizing culture were then subjected to RSGP analysis for identification.

As for L. fer~oxid~~m,spirilla were observed microscopically only in pyrite medium inoculated with environmental sediments (Table 2). However, bacilli, presumably T. ferrooxidnns, were also present in large numbers. When the cultures were transferred ta solid medium, L. ferrooridnns was not recovered.

The repeated plating and single-colony isolation technique could not be used.

Therefore, serial dilutions were done in 5% pyrite medium. However, growth of lep tospirilla was poor and O ther microorganisms were always present.

Consequently, an environmental strain of L. ferrooxidnns was not isolated.

Since isolation of L. Jrrrooxidntis from the environment proved to be problematic, the capacity of both type strains, CF12 and MK, to grow on different liquid and solid media was tested. Table 7 shows the results. One type culture,

CF12, showed growth in al1 media except TK medium, pH 3.0. The other type culture, MK, grew poorly in al1 liquid media and consequently was not used as a standard in RSGP analvsis. men these cultures were transferred to different iron salts solid media, results showed that no media supported growth (Table 7). Table 6. Gross colony characteristics of four shahs capable of oxidizing thiosulfa te

------Gross Mlb Mld M2aa M4b charac teris tics Size 0.4 - 0.7 mm 0.2 - 0.5 mm 0.3 - 0.4 mm 02- 0.4 mm Forrn circulai- circula r circular circular Eleva tion raised raised convex convex Margin en tire en tire entire entire white contour white Jvellow cen ter Density translucen t translucent translucent translucent Surface shiny, smooth shiny, smooth shiny, smooth shiny, smoo th Consis tency bu trous butyrous friable friable Table 7. Growth of Lrptospirillum ferrooxidn~zsMK and CF12 in different liquid and solid media

liquid medium grow th solid medium colony MK CF12 MK CF12

TK 3.3% FeS04 (2.1) - + ISP 0.3% FeS04 (3.0) - TK 3.3% FeS04 (1.8) + FeTSB (2.5) TK 3.3% FeS04 (3.0) - ISP 1.0% FeS04 (3.0)

Thiobncillris (2.5) + ~iiobncillils (2.5) pyrite (2.0) TK 3.3% FeS04 (1.8) TK 5.0% FeSO4 (1.8) ISP 3.3% FeS04 (3.0) -

Values within brackets represent the pH of the medium. Growth was defined as an increase in turbidity or the development of a rusty colorir. Since L. ferrooxihns showed poor growth in liquid medium, a higher temperature and the effect of shaking were tested. A pyrite culture enriched with sediments (Table 2) was used as an inoculum for three vessels containing

1% pyrite medium. Two cultures were incubated on a gyratory shaker, one at

30°C and the other at 22OC. The third culture was incubated at 22OC without shaking. The results are presented in Table 8. Growth did not occur in either culture that was gyrotated. However, a rust color was observed in the culture at

30°C. As for the culture that was not gyrotated, spirilla were observed rnicroscopically.

DNA purifia tion

Purification of DNA samples from E. coli 11303 was tested using four different extraction techniques: extraction with chloroform-isoamyl alcohol twice; extraction with phenol and then chloroform; extraction with phenol and then tw ice with diethyl ether; and, extraction with phenol-chloroform-isoamyl alcohol twice and then chloroform-isoamyl alcohol. The concentration and purity of DNA was rneasured by spectrophotometric analysis and the results are shown in Table 9. A DNA recovery of 46% was obtained with the extraction with phenol followed by a second extraction with chloroform, whereas recoveries using the other protocols were less than 5%. However, DNA purity as indicated by

OD,,/OD,, for the extraction with phenol followed by another extraction with diloroform was low. Extraction with only chloroform-isoamyl alcohol yielded the best puri ty. It should be noted tha t our purification protocol gave priority to Table 8. Incubation of a pyrite culture at different temperatures with or wi thou t shaking

medium tempera ture shaking growth

1%pyrite 1%pyrite Io/" pyrite Table 9. Optical density (OD) at different wavelengthç and purity of E. coli 11303 DNA recovered from 10 mL of culture using different extraction techniques

E. coli DNA sample OD at DNA ODat 260/280 260 nm recovery 280 nm

pure: two ex tractions with chloroform-isoamvl alcohol crude pure: extraction with p herlo 1, then with chloroform criide pure: extraction with phenol, b then twice with diethyl ether crtide pure: extraction with phenol- chloroform-isoamyl aicohol twice, then with chloroform- isoamyl alcohol DNA p~uityrather than DNA yield. Consequently, extraction using chloroform- isoamyl alcohoi only was chosen for DNA purification.

As for the RNase treatment, the results obtained by spectrophotometric analysis are presented in Table 10. The DNA recovery was higher for both samples without RNase treatment with recovery values over 50%. However,

DNA puritv was achieved bv induding RNase treatment. Since our protocol gave priority to DNA puritv, RNase treatment was included in the DNA isolation method.

A total of eight standards and 16 samples were used in RSGP analysis.

The raw data including DNA concentration and puritv for each are given in

Appendix 3, Table 1-5. DNA concentration and purity of the standards are presented in Table 1 (Apperidix 3). Table 2 (Appendix 3) includes the values for the controls and for the standards included in mixture A. The DNA concentration and purity of environmental wa ter samples enriched in different liquid media are listed in Table 3 (Appendix 3). Finally, Tables 4 and 5

(Appendix 3) present the values for five unidentified strains and for environmental samples witho~itenrichment in medium, respectively.

Values for DNA purity were approximately 1.8 for the majority of DNA samples. Two DNA samples, Glucose 2 and Glucose pH 2.3 (Appendix 3, Table

3), had low DNA concentration and purity. For DNA samples obtained directly from the environment (Appendix 3, Table 5), the puritv criteria was not met. Table 10. Op tical density (OD) at different wavelengths and purity of E. coli 11303 DNA recovered from 10 mL of culture with or without mase trea tment

E. coli DNA sample OD at OD at OD at DNA 260/280* 320 nrn 380 nm 260 nm recovery cnide pure: two extractions with chloro form-isoamyl alcohol crude pure: two extractions with chloroform-isoamyl alcohol pure: RNase treatment followed by iwo ex tractions with chloroform-isoamvl alcohol crude pure: RNase treatment followed by two extractions with chloroform-isoamyl alco ho1

'with background correction Three DNA samples had a 260/280 ratio below 1.8 and the other sample had a purity value above 1.8.

Ouantitative analysis of RSGP ~attems

Quantitative analysis of hybridization patterns was possible with the application of phosphoimaging plate technology. The photostirnulable luminescence values obtained for each standard in al1 the sarnples and the values used for quantitative analysis of hybridization patterns are given in Appendix 4.

Tables 1-8 of Appendix 4 show the quantitative analysis of the standards used as probes to define cross-hybridization and self-hybridization constants. Each table has the results for one probe, and the standard used as the probe is indicated in bold. Tables 9-26 of Appendix 4 represent the sarnples used as probes to define the fraction of standards present in the samples. Again, each table shows the results of one probe. Two samples, a sulfur-oxidizing culture and an environmental sarnple centnfuged to collect cells, were analyzed in duplicate.

Cross-hvbridiza tion

Al though self-hybridiza tion constan ts, given as k,/ k,, are calcula ted for every standard with every probe, only the ones that were determined when the standard and the probe were the same were applied to RSGP analysis of samples.

The constants for each of the standards are presented in Table 11. The degree of cross-hybridiza tion between standards was also determuied with quantitative analysis of hybridization patterns. Cross-hybridization data for al1 eight Table 11. Self-hybridization constants of standards spotted on the master fil ter

Standard kJk, T. ncidophilils ATCC 27807 T. nciriophiliis 64 T. nciriophiliis 46 T. fhioosidnns ATCC 19377 T.frrrooxidmis ATCC 23270 T.frrrooxi~ims FI T.frrrooxidms F2 L. ferrooxidnns CF12 standards are displayed through Fig. 5 to Fig. 12. In general, cross- hybridizations were low relative to self-hybridization, which was taken as 100%.

The type strain of T. ncidophilus showed slight hybridization with both environmental isolates (Fig. 5). However, significant cross-hybridization occurred between both environmental isolates of T. ncidophillis (Figs. 6 and 7).

Also, both T.ferrooxi~inns environmental isolates had substantial degrees of cross- hybridization and showed weak cross-hvbridization tvith the tvpe strain (Figs. 10 and 11). DNA from T. thioowidnns and L.frrrooxi~imsappeared to be unique (Figs.

8 and 12).

Analvsis of controls bv RSGP

After the self-hvbridiza tion constants were determined, the mas ter filter was probed with sampLes to measure the fractions of a given standard in the samples. The f, values, calculated without correction for cross-hybridization, were plotted against standard letter. The results show that both controls hybridized with al1 the standards. Mixture A consisted of sirnilar DNA amounts for each standard except T. ferrooxidnm FI (Table 3). Figure 13 shows strong hybridization occurring with al1 strains of T.ncidophillis and one environmental strain of T.forooxidnns. The second control was a mixture of standards cells from which DNA was extracted. Again, ail T. ncidophilus standards and one environmen ta1 strain of T. frrrooxidnris showed slightly more hvbridiza tion with mixture B (Fig. 14). In dl, results for both controls were similar except a weaker hybridization pattern was observed with mixture A. C D E F standards

Figure 5. Cross-hybridîzation of T. ncidopliiiils ATCC 27807 (A,T. ncidophilils ATCC 27807; 0, T. ncidophilus 64; C, T.ncidophilils 46; D, T. thiooxisims ATCC 19377; E, T.ferrooxidn12s ATCC 23270; F, T. ferrooxidm~sFI; G, T.ferrooxidnns F2; H, L. ferrooxidans CF12) C D E F standards

Figure 6. Cross-hybridization of T. nciriophiltls 64 (A, T. ncidoplzilils ATCC 27807; B, T.ncirlophilzis 64; CrT.aciduphiliis 46; D, T. thiooxidnns ATCC 19377; E, T.ferrooridiz~is ATCC 23270; F, T.ferrooxidntzs FI; Gr T.ferrooxidrins F2; H, L.ferrooxidrins CF12) standards

Cross-hybridization of T. ncidophilirs 46 (A, T.ncihphil~~s ATCC 27807; B, T. ~cidophilr~s64; C,T.acidophifiis 46; D, 7'. tliiooxidtzns ATCC 19377; E, T.ferrooxidnm ATCC 23270; F, T.ferrooxirlnns FI; G, T.frrrooxidmzs F2; H, L. ferruoxidans CF12)

C D E F standards

Figure 10. Cross-hybridization of T.ferrooxidans FI (A,T. ncidophiltls ATCC 27807; B, T. ~cidophiilis64; C, T.ncidophilzis 46; D, T. thiooxidmzs ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T.ferrooxicinns FI; G, T.ferrooxihns F2; H, L. fe~rooxidnnsCF12) C D E F standards

Figure 11. Cross-hybridiza tion of T.ferrooxidnns F2 (A, T. ncidophilirs ATCC 27807; 8, T. ncidophiliis 64; C, Tmidophilils 46; D, T. thiooxicinns ATCC 19377; El T.ferrooxidrzns ATCC 23270; F, T.ferrooxidnns FI; G, T.ferrooxidnns F2; H, L./errooxidmzs CF12) standards

Figure 12. Cross-hybridization of L. jérrooxidms CF12 (A, T. ncidophillîs ATCC 27807; 8, T.aciciophilzîs 64; C, T.acidophilzis 46; D, T. thiooxidnns ATCC 19377; E, T. ferrooxidnns ATCC 23270; F, 7'. ferrooxidnns FI; G, T.ferrooxidnm F2; H, L. ~errooxidnnsCF13 D E F standards

Figure 13. RSGP of rnixhire A of standard genomes (A,T. nciclophiltis ATCC 27807; B, T. ncidophillis 64; C, T.~cidophilirs46; D, T. thiooxihns ATCC 19377; E, T.ferrooxiifnns ATCC 23270; F, T.ferrooxzdnns FI; G, T.ferrooxidms F2; H, L. fmrooxidnns CF12) A B C D E F G H standards

Figure 14. RSGP of mixture B of standard genomes (A,T. ociduphilzis ATCC 27807; B, T. ncidophiliis 64; C, T.ncidophiliis 46; D, T. thiooxidnns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T.ferrooxidaris FI; G, T.ferrooxihns F2; H, L. ferrooxidnns CF12) RSGP analvsis of environmental sarnvles after enrichment

Figures 15 through 20 show the application of RSGP in the identification of microorganisms in acid mine drainage samples after enrichment. The water sample collected was used to inoculate directly or indirectly seven liquid media, including TK, tetrathionate, Shrkey's Msdium 1 and glucose. Following growth and DNA extraction, six DNA preparations were obtained for RSGP analysis.

Enridiment in TK medium favored the growth of environmental strains of

T.prrooxicfczns (Fig. 15). T. thiooxidnns was the dominant isolate in Starkey's medium 1 (Fig. 16). DNA from the other standards showed weak hybridization, except L. ferrooxidnns which showed no hybridization. Following enrichment in tetrathionate medium, the DNA sample hybridized with al1 the standards except

L. Jerrooxidms (Fig. 17). Again, this lep tospirillum showed no hybridization.

Standard T. thiooxihns was the dominant isolate in that sample. Growth in glucose followed by growth in tetrathiona te resulted in the presence of al1 strains of T.ncidophilris (Fig. 18). Enrichment in glucose did not seem to have any of the standards represented in any significant quantity (Fig. 19). However, lowering the pH of the glucose medium gave similar results to growth in glucose followed by grorvth in tetrathionate. The DNA sample showed hybridization with al1 strains of T. ncidophillls (Fig. 20).

Analvsis of unidentified cultures bv RSGP

Four strains capable of oxidizing thiosulfate were also isolated from a mixed culture via single colony formation. Following DNA extraction, RSGP standards

Figure 15. RSGP of DNA extracted from an environmental sample enriched in TK medium (A,T. ncidophilus ATCC 27807; 8, T. midophilus 64;C, T.ncidophilus 16; D,T. thiooxidnns ATCC 19377; E, T.fevooxidons ATCC 23270; F, 7'. ferrooxidnns FI; G, T.fermxidnns F2; H, L. ferrooxicinns CF12) C D E F standards

Figure 16. RSGF of DNA extracted from an environmental sample enriched in Starkey's medium 1 (A, T. acidophilns ATCC 27807; B, T. ncidophilris 64; C, T.ncidophiliis 46; D, T. thioowihns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T.ferrooxidnns Fi.; G, T.ferruoxidnns F2; H, L. ferrooxicinns CF12) A B C D E F G H standards

Figure 17. RSGP of DNA extracted from an environmental sample enriched in tetrathionate medium (A, T. ncidophilus ATCC 27807; B, T. ncitioplzilr~s63; C, T.ncidophi1iis 46; D, T. thiooxidms ATCC 19377; E, T.ferrooxidms ATCC 23270; F, T.firrooxidatis FI; G, T.ferrooxidnns F2; H, L. ferroowidms CF12) A B C D E F G H standards

Figure 18. RSGP of DNA extracted from an environmental sample enriched in glucose medium which was then transferred to tetrathionate medium (A,T. acidophilzis ATCC 27807; B, T. ncirlophillis 64; C, T.ncidophiliis 46; D,T. thiooxidnns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F,T. ferrooxidnns FI; G, T. ferrooxidmzs F2; H, L. ferrooxidnns CF12) 1 I I I 1 A B C D E F G H standards

Figure 19. RÇGP of DNA extracted from an environmentai sample emiched in glucose medium (A, T. ncidophiliis ATCC 27807; B, T. ncidoplziliis 64; Cf T.nciduphilrls 46; D, T. thiooxicinris ATCC 19377; E, T.ferrooxidntts ATCC 23270; F, T.Jerrooxidnns FI; G, T. ferrooxidnns F2; Hf L. fmoxidnns CF12) analysis was used to identify these strains. Ali four DNA samples hybridized strongly with al1 isolates of T. fmooxidnns (Fig. 21-24). T.thiooxidnm also showed hybridization but its fractions were less than 0.1 in all the samples. Following such results, the four strains were tested for their capauty to oxidize ferrous iron.

Only one strain showed growth in TK medium.

As for the strain isolated by serial dilution in Starkey's Medium 1, RSGP analysis showed tha t T. tliiooxidntis was dominant (Fig. 25-26). However, weak hybridization was observed with T. ncidopkil!ls (0.1 or less) and T.frrrooxidnris (0.1 or less). This culture was analyzed twice and the resulting hvbridization pa ttems were sirnilar .

Analvsis of microbial communities bv RSGP

To determine the composition of microbial communities in acid mine effluents, water samples and sediments were collected and analyzed by RSGP.

DNA frorn cells collected by centrifugation of a water sample hybridized weakly with al1 the standards (Fig 27-28). This DNA sample was analyzed twice and the resul ting hybridiza tion patterns were similar. However, T.ferrooxidnns was dominant. As for the water sample that was prefiltered and centrifuged, the dominant standard was T. ncidophilus (Fig. 29). T.ferrooxicinns also showed hybridization but not as strongly as T.ncidophilirs. Little hybridization resulted from the DNA sample obtained from sediments (Fig. 30). T.ferroowidms showed the most hybridization with a 0.02 fraction. A B C D E F G H standards

Figure 21. RSGP of DNA extracted from a thiosulfate-oxidizing s train Mlb (A, T. ncidophil lis ATCC 27807; 8, T. ~cidophihs64; C,T.nciiiophi1zis 46; D, T. thiooxidnm ATCC 19377; E, T. ferrooxzdnns ATCC 23270; F, T.ferrooxidmzs FI; G, T.fêrrooxidmzs F2; H, L. fmooxidnns CF12) A B C D E F G H standards

Figure 22. RSGP of DNA extracted from a thiosulfate-oxidizing shah Mld (A, T. cid do phi lus ATCC 27807; B, T.~cidophihs 64; C, T.acidophiliis 46; D, T. thiooxidnns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T.ferrooxidnns FI; G, T.frrrooxidnns F2; H, L. ferrooxidrrns CF12) C D E F standards

Figure 23. RSGP of DNA extracted from a thiosulfate-oxidizing shah M2aa (A,T. izcidophiiris ATCC 27807; B, 7'. ncidophillrs 64; C, T.ncidophilils 46; D, T. thiooxidms ATCC 19377; E, T.ferruoxidnns ATCC 23270; F, T. j'ierrooxih~isFl; G, T.fmooxidnns F2; H, L. ferrooxihns CF12) C D E F standards

Figure 24. RSGP of DNA extracted from a thiosulfate-oxidizing strain M4b (4,T. ncidophiliis ATCC 27807; B, T. acidophilirs 64; C, T.acidophiliis 46;D, T. thiooxihns ATCC 19377; E, 7'. ferrooxido~rsATCC 23270; F, T.ferrooxirinns FI; G, T. fmoxidons F~;'H,L. fmooxidans CF12) A B C D E F G H standards

Figure 25. RSGP of DNA extracted from a sulfur-oxidizing culture B (analysis 1) (A,T. ncidophiliis ATCC 27807; B, T. ncidophiliis 64; C, Tmidopliilzis 46; D, T. thiooxidnns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T.ferrooxidnns FI; G, T. fmooxidnns F2; H, L. fmooxidans CF12) C D E F standards

Figure 26. RSGP of DNA extracted from a sdfur-oxidizing culture 0 (analysis 2) (A,T. ncidophiliis ATCC 27807; B, T. nciciophil~is64; C, T.ncidophiliis 46; D, T. thiooxidnns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T.frrrooxidnns FI; G, T.fmooxidnns F2; H, L. fmooxicinns CF12) standards

Figure 27. RSGP of DNA extracted from an environmental water sample centri fuged to collec t cells (analvsis 1) (A, T. nci~iophilils ATCC 27807; 8, T. ncidophilirs 64; C, ~.&do~hilirs$6; D, T. fhiooxidn~zs ATCC 19377; E, T.fmoowidnns ATCC 23270; F, T.fvrrooxidms FI; G, T. ferrooxidrrns F2; H, L. fwooxicinns CF12) C D E F standards

Figure 28. RSGP of DNA extracted from an environmental water sample centrifuged to collect cells (analysis 2) (A,T. ~cidophiliisATCC 27807; 8,T. acidophilt~s64; C, T.ncidophi2rls 46; D, T. thiooxidnns ATCC 19377; E, T. frrrooxidans ATCC 23270; F, T.j2rrooxidnm FI; G, T.ferrooxidnris F2; H, L. ferrooxidnns CF12) A B C D E F G H standards

Figure 29. RÇGP of DNA exhacted from an environrnental wa ter sampie prefiltered and centrifuged to collect cells (A, T. ncidophiliis ATCC 27807; B, T. acidophihs 64; C, T.ncidophilris 46; D, T. thiooxidms ATCC 19377; E, T.ferruoxidnns ATCC 23270; F, T.ferrooridnns FI; G, T.ferrooxidmis F2; H, L. frrrooxihns CF12) r-Ir I I I I II A B C D E F G H standards

Figure 30. RSGP of DNA extracted from an environmental sediment sample (A,T. ncidophilils ATCC 27807; B, T. nciiiophilus 64; C, T.ncidophiliis 46; D, 7'. thiooxidnnç ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T.frrrooxidnns FI; G, T.fmooxidnns F2; Hf L. ferrooxidnns CF12) DISCUSSION

DNA isolation roto col

DNA extraction and purification from microbial cells, especially

Thiobncill ils spp. and other environmental isola tes, can be difficult and problematic. Often, protocols are s~utablefor some studies but not for others. In this study, RNase treatment and chloroform-isoamyl alcohol extraction were found to improve DNA purification. However, in accordance with Fuhrman et al. (1988), the elimination of RNase treatment improved the recovery of DNA.

Since the protocol was developed in an effort to ensure purification of DNA samples rather than give a high yield, RNase treatment was included in the purification of DNA. However, to improve DNA yield in further studies,

Fuhrman et nl. (1988) suggested centrifiiging cellular debris suspended in TE buffer with SDS at 12 to lj°C since SDS precipitates at PC, trapping much of the

DNA.

Lysozyme was also included in the protocol. However, its effect was never tested. Since initial work on DNA extraction showed poor ce11 lysis, lysozyme and freeze-thaw cycles were always included to improve lysis.

However, Fuhrman et al. (1988) found lysozyme to be unnecessary for bacterial lysis and to increase the protein content which made purification more difficult.

This finding should be taken into consideration in future DNA isolations. Isolation of T. thiooxidnns

Isolation of T. thiooxidnlzs using hosulfate or tetrathionate as sources of energy for growth was not successful. It seems that T.ferrooxihns outcompeted

T. thiooxidmzs on thiosuifate solid medium. Colonies of T. thiooxidarrs on solid medium are very tiny, which can account for its unsuccessful isolation.

However, the use of a sulfur medium resulted in the successful isolation of a strain of T.thiooxidnns. As T. tliioowidnizs possesses the abilitv to oxidize elemental sulf~lrrapidly (Kelly and Harrison 1989), this can explain why isolation on sulfur was sticcessf~ll.

Isolation of L. f~rrooxidms

Isolation of L. ferrooxidnris can be very difficult. Difficulties have been reported in growing this iron-oxidizing acidophilic bacterium on most solid media (Johnson and McGinness 1991; Norris and Kelly 1982). Harrison (1984) and Goebel and Stackebrandt (1994a) failed to obtain colonies of L. ferrooxidnns on solidified media. Like T.fwooxidntis, L. ferrooxidn~is is very sensitive to organic matter, including small quantities of sugars present as impurities in agarose (Harrison 1984; Johnson 19%).

A nurnber of alternate methods have been used with some success.

Highly purified agars were used and bacteria grew poorly because some of the sugar molecules in the gelling agent were subject to hydrolysis in acid solution and the released sugars inhibited ce11 growth. The most successful method was the double-layer plate technique described by Johnson and McGimess (1991). The first layer consists of a mixture of freshly-grown audophilic heterotrophs

(Acidiphilirmz sp.) and a ferrous iron/tryptone soya broth agar medium. The

second layer of sterile inorganic medium without the heterotrophs is poured on

top of the set gel. The introduction of heterotrophs reduces the concentration of

sugars and metabolic waste products, allowing chemolithotrophs to grow. This

double-laver plate technique was not used in this project because an acidophilic

heterotroph capable of growth in a ferrous iron/ hyptone soya broth/galactose

medium couid not be obtained. Growth in such a medium was necessary to

adapt the heterotrophic bacterium to the chemical conditions within the solid

medium (Johnson 1995).

Isolation of L. frrrooxidnw from mixed cultures can also be a difficult

procedure. Its minimum doiibling time is much higher than that of

T.ferrooxidnns (Norris and Kelly 1982). In a recent review article, Rawlings et ni.

(1999) explained that, in liquid ferrous iron or pyrite medium, T.firrooxidnns will

initiaily outgrow L. firrooxicims because low amounts of ferric iron are present.

T.ferrooxicfnns is able to build up large numbers of cells before conditions become more favorable for L. ferrooxidn,is, which can eventually dominate because of its greater affinity for ferrous iron and its greater resistance to femc iron. Also, the faster growth rate of T. ferrooxidms results in the elirnination of L. ferrooxidîzns in rnixed cultures subcultured in ferrous iron medium (Walton and Johnson 1992).

This can explain the large numbers of bacilli in the pyrite medium and the lack of success with serial dilution. Battaglia et nl. (1994) were able to obtain a

Lrptospirillilm-domimed culture by successive enriclunent in ferrous iron medium containing femc iron. Also, a prolonged incubation penod might have been required for the isolation of L. ferrooxidnns by serial dilution. Noms et al.

(1988) isolated a Leptospirillum-like bacterium from a mixed culture via single colony formation on ferrous iron agar following serial culture for three years.

Another factor to consider when favoring the presence of L. ferrooxihizs is the temperature of the habitat. It seems that at temperatures above 20°C,

L. frrrooxidms has generation times similar to those of thiobacilli. However, when temperatures become lower than 20°C, the genera tion times of leptospirilla increase more than those of T.frrrooxidniis (Hallman et ni. 1993; Sand et al. 1992).

All environmental samples had temperatures below 20°C aside from the sediment sample (28.1aC)enriched in pyrite, which was the only culture to carry lep tospirilla.

The maintenance of L. ferrooxidnns in liquid medium can be a challenge.

When maintained in liquid ferrous iron medium, it was found that L.férrooxihns requires frequent subculturing (Norris and Kelly 1982). In this project, the effect of shaking and a higher temperature were tested. It seems that the growth of

L.ferrooxidms was favored by the absence of shaking and that a higher temperature did not support its growth. However, a rust color was observed in the culture incubated at 30°C,which indicates the occurrence of iron oxidation.

No cells were observed. Thus, the higher temperature must have caused the oxidation of ferrous iron. Overall, the isolation of L. ferrooxicinns from mixed cultures and its cultivation proved to be problematic. RSGP analvsis

Several studies have been published in the last two decades on the genetic diversity of the genus Thiobncillirs. Yates and Holmes (1986b) showed that whole genome probes could be used to identifv strains of 7'. ferrooxidnns in the presence of T. ncidophilirs and T. novellrls. In another study, different strains of

T. thioorihns showed negligible DNA-DNA homology with numerous strains of

T. ferrooxihns (Harrison 1982). The lack of homology among genomes of

Thiobncill ils sp. can, therefore, be exploi ted in RSGP analysis.

Since many reports indicate a high degree of genornic diversity among strains of T.ferrooxi~inrzs (Harrison 1982; Lane et ni. 1992; Novo et nl. 1996), environmental strains and type cultures of T. frrrooxidnm and T. ncidophilris were spotted on the master fil ter. Ideally, environmental strains of L. ferrooxidnns and

T. thiooridnns should also have been present on the master filter. However, their isolation frorn acid mine drainage environments was unsuccessful. RSGP analysis did distinguish between environmental strains and type cultures of

T. frrrooxidnns and T. acidophilits. However, strong cross-hybridization was observed among environmental strains of the same species, probably because they occupied the same habitat. Standards should show little or no cross- hybridization (Voordouw et al. 1991). In this study, cross-hybridization between the standards was tolerated. Further RSGP analyses could be improved by elimina ting cross-hybridiza tion. The genomes of bo th environmental strains of

T.fmooxidnlrs could be combined and used to spot the filter as a single standard.

The same could be done for both environmental strains of T. acidophilns. The success of RSGP analysis is largely dependent on the master filter

produced, in other words, the number and types of bacterial standards spotted

on the filter. Bacteria expected to be found in some of the samples should be

spotted on the filter as standards. These can be obtained by isolation from the environment or they can be obtained from type culture collections. Once a

master filter is produced, strains isolated from the environment and purified can be identified by RÇGP analysis. In this study, four thiosulfate-oxidizing strains

were identified as T. frrrooxi~imisand one sulfur-oxidizing culture was identified as T. fhiomidms. Since an environmental strain of T. tlzioosidnns was absent on

the filter and its genome has a low degree of cross-hybridization with the type culture, this culture could be incorporated on the filter as a standard after colony

purification.

As for the nurnber of bacterial standards spotted on the filter, only a small

fraction of the bacterial population found in acid mine drainage environments is present, whidi is obviously not representative. Since it was not known if RSGP analysis could be used to characterize the bacterial population in acidic environments, only four species, the most studied in such environments, were chosen for RÇGP analysis. Now that it is known that RSGP analysis can be used

to study such environments, this method can be improved by expanding the mas ter fil ter to include a varie ty of heterotrophic bac teria su& as Acidiphiliitrn spp ., and other Thiobncill us species. RSGP analvsis of controls

As a test, two mixtures of standard genomes were analyzed by RÇGP. For mixture A containing equal DNA amounts (excluding T. ferrooxidnns FI), stronger hybridization was observed with strains of T. ncidophilrrs and

T. frrrooxidnns. The same was noted for the hybridiza tion pattern obtained wi th mixture B, which initiallv contained equal numbers of cells. Ideally, the calculated fX values should be equal for al1 standards. However, hybridization was present among standards of the same species. Also, differential labeling of h and the chromosomal sample DNA, or pipetting errors when standard and h DNAs are combined, can result in different fraction values.

RSGP analvsis of DNA samdes after enrichment

RSGP analysis of samples af ter enrichmen t provides information on the bacterial species present in that environment, and not on their numbers, since enrichment shifts the community composition away from that present in the environment. It also shows which bacterial species are being selected by different culture media. These results show that TK medium favored the growth of T.ferrooxidnrts, the only thiobacillus to oxidize ferrous iron. L. ferrooxidnns was not identified in this medium. Similarly, Pizarro et al. (1996) showed that

T. ferrooxidnns becomes the dominant iron-oxidizer after growth in ferrous iron medium. In a study of microbial diversity in uranium mine waste heaps,

L.Jmoxidnns was detectable only in assays with pyrite as the substrate

(Schippers et ni. 1995). The lack of growth of L. ferrooxidnns in ferrous iron medium can be explained by the inhibition of its growth at high ferrous iron concentrations and the faster growth rate of T.ferrooxidnns. These results suggest that L. ferrooxidmzs could be more common in acid mine drainage environments than previously estimated by cultural analysis in ferrous iron medium. Other explanations for its absence will be discussed later.

It was interesting but not surprising to observe that sulfur selected for

T. thiooxidrzris. This selection was observed earlier in this study when the culture obtained from serial dilution in suifur was identified as T.thiooxi~imis. Its abiliîy to oxidize sulfur was also observed when T. tkiooxiiimis populations were 2 to 3 orders of magnitude greater than T.ferrooxidmis at an elemental sulfur deposit in

Yellowstone National Park (Nordstrom and Southam 1997). Konishi et al. (1995) found that the specific growth rate for T. thiooxi~imison elemental sulfur was 2.58 da y-', w hich is higher than that of T.frrrooridnns es timated a t 0.5 day-' by Espejo and Romero (1987). Sulfide rninerals are the major natural substrates for

T. ferrooxidmzs, and growth on elemental sulfur in the laboratory may place certain pressures and restrictions on ce11 me tabolism.

Enrichment in tetra thiona te medium also favored the growth of

T. thiooxihns. The oxidation of tetrathionate yields sulfur among other reduced sulfur compounds (Pronk et d.1990a; Suzuki 1974). Therefore, the presence of sulhr selected for T. t/iiooxicimis.

The DNA sample obtained from the glucose medium hybndized slightly with al1 strains of T. ncidophilils. Such minimal hybridization was probably because of the large number of yeasts supported by this medium. As a result, for subsequent enrichment, the pH of the medium was lowered from 3.2 to 2.3.

RSGP analysis showed that a lower pH favored the growth of T. ncidophilris, since most eucaryotes can not tolerate such low pHs. Also, an inoculum from the glucose medium was inhoduced into a tetrathionate medium to favor the growth of T. ncidopkilils. As expected, the DNA sample showed hybridization with all strains of T.ncidophilrls.

RSGP analvsis of DNA sam~lesfrom the environment

Ini tially, environmental samples were analyzed af ter liquid culture enrichment. RSGP following enrichment made qualitative identification possible. In other words, bacterial standards present in liquid enrichment cultures were identified. However, enrichment of environmental samples in ciifferent media resulted in the separation of the individual genomes from the target environment. Thus, the RSGP patterns described the microbial community in terms of its culturable component and did not represent the difficult- to-cul hm or non-culturable componen t. To describe the communi ties as they existed in the native environment, unchanged by enrichment, RSGP analysis was done using DNA extracted directly from cells recovered from liquid and solid environmental samples.

The DNA sample obtained from cells collected by centrifugation hybridized weakly witli al1 the standards exduding L. ferrooxidnns. However,

T.ferrooxidnns was dominant. It is known that T.ferrooxidnns is abundant in acid mine drainage environments since it can oxidize ferrous iron and inorganic sulfur compounds. This sulfur-containing environment should be energetically favorable for T. thiooxidnns and may explain its association with T.ferrooxidans

(Nordstrom and Southam 1997). It is also known tha t T. ncidophilils iç often associated with T.fewooxidnns. Since T.ncidophilzts is oligotrophic, it can thrive on low concentrations of organic compounds excreted from T. ferrooxidans.

The weak hvbridization pattern obtained after centrifugation of the water sample may have been caused by the presence of eucaryotes such as fun@ and algae. Therefore, a second water sample was prefiltered to rernove eucaryotes and then centrifuged to collect cells. In this case, the hybridizations were s tronger. The dominant bac terium was T. ncidophil ils, especially the type strain.

T.ferrooxirlmts also showed hybridization but not as strongly as 7'. ncidophil~is.

This result is congruent with the observation of McGinness and Johnson (1993), who found tha t acidophilic heterotrophic bacteria domina ted several acid mine drainage samples. It is possible that the conditions of the habitat were more favorable for T. ncidophilils growth. In uranium mine waste heaps, moderately acidophilic thiobacilli such as T. ~irnpolitnnlts,T. interntrdizrs, and T. novcllris were found in higher number than was T.ferrooxidans, in ore samples with neutral pH

(Schippers et n2. 1995). These results indicate that the substrate needed for

T. acidoph il lis could no t origina te from pyrite oxida lion by T. ferrooxidms.

Schippers et al. (1996) showed tha t thiosulfa te, tri thionate and tetra thionate are key in terrnedia te sulfur compounds in oxida tive pyrite degrada tion. These compounds are known to be suitable substrates for T. ncidophilrls. Moreover, being capable of heterotrophic growth, 7'. ocidophilus can oxidize organic compounds provided in water drainage and in ce11 material released from autotrophic bacteria through ce11 leakage and death, and perhaps in association with pyrite (Harrison 1984). T.ferrooxidnns can feed T. ~cidophihsby the excretion of organic compounds (Hallman et al. 1993; Tuttle and Dugan 1976;

Wichlacz and Thompson 1988). Also, the fact that T.ferrooxidnns is known to adhere to the surface of its substrate could explain the dominance of

T. ncidopliiliis. Prefiltration may remove particles on which T.fe'rrrooxidnns was attached, reducing the number of cells in the water sample.

Concentrating the bacteria by centrifugation was generally unsatisfactory since the procedure was time-consuming and a large volume of water was needed. As a result, bacteria were collected on a nitrocellulose filter membrane, after prefil tra tion to remove eucaryotes. However, the DNA sample ob tained from cells collected on the nitrocelli~losemembrane was contaminated with material extracted frorn the filter during processing. It is possible that this material originated from isoamvl alcohol dissolving some component(s) in the nitrocellulose membrane. Thus, this DNA sample could not be analyzed by

RSGP. A fluorocarbon-based filter could be used since it would not interfere with DNA extraction and purification.

In an attemp t to detect L. ferrooxidnns, cells were removed from sediments by mechanical shaking. Little hybridization resulted from the DNA sample obtained from these cells. It is possible that the la& of hybridization was due to the method used for ce11 collection. Unsuccessful attempts were made by Goebel and Stackebrandt (1994a) to isolate bulk genornic DNA directly from washed ore samples.

Many methods for extracting DNA from soi1 bacterial communities incorporate polyvinylpolypyrrolidone (PVPP), which removes humic acid contaminants by adsorption to this insoluble polymer. The polymer PVPP was not included in ths project. The low concentration of organic material in acid mine drainage environments indicates that potential DNA interfering substances such as humic auds are absent (Goebel and Stackebrandt 1994a).

In other reports, blenders are used instead of mechanical shaking. The use of blenders has been shown to ameliorate the separation of the bacterial fraction from soi1 (Holben rt al. 1988). Blending was not used because the sediments were too large in particle size.

The absence of L. ferrooxidms in environmental sam~les

The failure of a standard to appear in an environmental sample does not necessarily mean that it is absent from that site. Several factors such as the collection of cells and the abiotic conditions of the habitat can play an important role in the detection of a speafic standard.

One factor to consider when isolating L. firrooxirlnns is its great capacity for attachment to substrate (Harrison and Norris 1985; Norris et RI. 1988).

According to Sand et RI. (1992), sediment samples should be shaken for 2 h to detach cells since L. fevooxidmrs cells adhere more tightly to surfaces than do cells of T.ferrooxidnns. This could explain the presence of 7'. ferrooxidnns and the absence of L. ferrooxidnns in the sediment sample since the sediments were incubated for only 1 h on a rotary shaker.

The temperature of the habitat is another factor to consider when favoring the presence of L. ferrooxidnns. As previously stated, at temperatures lower than

20°C, L. ferrooxidms has generation times higher than those of T.ferrooxidnns. The sediments were collected at the same time as the water samples that underwent prefiltration, and these samples had a temperature of 1°C. Furthermore,

L.ferroo+i~inns was never detected in any water samples analyzed by RSGP.

Similariy, Hallman et nt. (1993) and Sand et nt. (1992) were never able to detect leptospirilla in any mine water samples.

Conclusion

RSGP was developed in order to analyze bacterial communities in environments where they are poorly characterized and/or difficult to grow

(Voordouw et nl. 1991). Such is true of the microbial cornmunities found in acid mine environments, and it was shown that RSGP can be used to identify such microorganisms. Although this method is fast, and sensitive and provides identification of multiple rnicroorganisms in a single s tep, i t camot dis tinguish between live and dead cells in an environmental sample without enrichment.

Aside from this problem, RSGP is a highly convenient and sensitive method for the identification of rnicroorganisms found in acid mine drainage, avoiding isolation and purification of bac teria, which is difficult and time-consuming. This method could have many applications in acid mine environments.

For example, a comparison of KGF patterns obtained at different intervals

throughout a year could help in understanding the role of specific bacteria in

acid mine drainage. Also, a better understanding of the effectiveness of different

treatments on acid mine drainage could be obtained when RÇGP analysis is done

periodically on samples obtained from treated acid mine effluents. Moreover,

RSGP could be used to characterize and /or moni tor bac terial populations in

bioleaching processes. The use of RSGP analysis has many advantages and its

application to acid mine environments should allow the identification and

characterization of bacterial communitieç in such environments.

The purpose of the current study was to isolate environmental strains of

T.ferrooxidnns, T.ncidophilris, T. tlziooxidnns, and L. ferrooxidnns and to apply RSGP

analysis to the identification of bac teria recovered from acid mine effluents.

Although RSGP analysis has been used to identify sulfate-reducing bacteria

recovered from water samples affected by acid mine drainage (Telang rt al. 1994), iron- and/or sulfur-oxidizers have never been spotted on the master filter. The da ta in this s tudy support the following conclusions:

1. Leptospirillrim ferrooxinnns was not recoverable on iron salts solid medium.

2. DNA purity was obtained with mase treatment and two chloroform-

isoamyl alcohol extractions. Environmental strains of the same species showed strong cross- hybridiza tion.

Cross-hybridization between type s trains and environmental s trains was no t significan t.

Iron salts medium supported the growth of T.frrrooxidnns only.

Sulfur medium favored the growth of T. thiooxidnris.

Growth in glucose followed bv growth in tetrathionate resulted in the selection of 7'. ncidophil lis.

Reverse sample genome probing analysis of an environmental sample wi thout enrichment in culture media yielded a be tter hybridization pattern w hen prefil tered.

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Yates, J.R., Lobos, J.H., and Holmes, D.S. 1986. The use of genetic probes to detect miuoorganisms in biomining operations. J. Ind. Microbiol. 1: 129-135. APPENDIX 1: CULTURE MEDIA

Table 1. Iron salts (TK) medium (Tuovinen and Kelly 1973)

Solution A

Adjust the pH to 2.5 with H2S0,. Autoclave at 121aC for 20 min. Allow to cool down to room temperature.

Solution B

Sterilize by membrane filtration. Divide the solution in 200 rnL bottles. Add one bottle to solution A and mix well. This medium is slightly modified since the final is pH 2.1 instead of 1.6. Table 2. Tetrathionate medium - Formulation used by Dr. O.H. Tuovinen, Ohio State University

Liquid medium Solution A m,Po, 3.0 g/L H,O (NH,)2SO, 3 .O MgSO, UFI,O 0.5 c~cI,~~H@O .25 WO, 3.2 Adjust the pH to 3.8.

Solution B Na, EDTA Z~SO,@7H,O MnC1, @~H;o ~0~1~;6~~0 (NH,),Mo;O, FeSO, -7H20 CuSOp5H20

Adjust the pH to 3.8. This solution is stable for two weeks after its preparation. Add 10 mL of solution B to solution A. Mix well. Adjust the pH of the mixture to 3.2. Sterilize by membrane filtration.

Solid medium

Sohtion A same as previous excep t that the volume is 800 mL HzO instead of 1 L

Adjust the pH to 3.8.

Sol~itionB same as previous

Adjust the pH to 3.8. Add 10 mL of solution B to solution A. Mix well. Adjust the pH of the mixture to 3.2. Sterilize by membrane filtration. Allow the mixture to equilibrate in water bath at 50°C.

Solution C agarose 8 g/200 mL H20

Autoclave at 121°C for 20 min. Allow solution C to cool down in water bath. Add slowly the mixture to solution C. Mix and pour in plates. Table 3. Starkey's medium 1 (Chan and Suzuki 1993)

Adjust the pH to 2.3 with KSO,. Sterilize by membrane filtration and inoculate. Spread 50 g of p&vdered çulfur evenly on the surface. Table 4. Iron salts purified (ISP) agarose medium Mishra et al. (1983) modified the medium of Manning (1975)

Solution A

Adjust the pH of distilled water with $O4 before adding FeSO,VH,O. Sterilize bv membrane filtration. Divide solution A in three bottles of- 300 mL.

Solution B WH,)SQ 0.165 g/550 mL H20 MgSO, a7H20 0.275 KCL 0.055

Adjust the pH to 3.0 with HISO,. Autoclave at 1210C for 15 min.

Solution C

agarose 6 g/150 rnL H20

Autoclave at 1210C for 15 min.

AIIow solutions B and C to cool down in water bath at 55T for several minutes. Add a bottle of 300 mL of solution A to solution B and mix well. Heat slightly in water bath and add slowiy the mixture to solution C. Mix gently. Pour in plates. The final pH of the medium is 3.0. Table 5. Medium S (Postgate 1966)

A. Liquid medium

5.0 g/L H20

MgSO, e7H,O C~CI,Q&O FeSO, 0-0

Adjust the pH to 4.0 with -0,. Sterilize by membrane filter.

B. Solid medium

Solution A Na2S20,*5H20 5.0 g/700 mL H,O KH, PO, 4.0 (N%):SO.I 4.0 MgSO, UH,O 1.O24 C~CI,*2.,0 0.331

Adjust pH to 4.0 with H,SO,. Sterilize bv membrane filter. Heat slightly in water bath.

Solution B agarose 15 g/300 mL H,O

Autoclave. Let solution B cool down for a few minutes in a water bath. Add solution B gently and mix slowly. Pour in plates. Table 6. Waksman medium (Rodina 1972)

ColIoidal sdphur 10.0 g/L -0 KH,PO, 3 .O WH,) -0, 0.2 MgSO, @-O 0.5 CaClz*2H20 0.126

The pH is adjusted to 3.0 with H,SO,. Sterilize bv tyndalisation. Table 7. Thiobncillzis medium B (ATCC medium 238)

Adjust the pH to 4.1 with H?SO,. Autoclave. Table 8. Pyrite medium

4% K,HPO, 10.0 mL il~Mgso,.-O 10.0 4% (NHJ $O4 10.0 H20 770

Adjust the pH to 2.0 with H,SO,. Autoclave at 121°C for 20 minutes. Allow to cool down to room temperature. Dispense aseptically 100 mL aliquots in 250-mL Erlenmeyer flasks. Add 5 g of pyrite to each Erlenmeyer flask. Table 9. FeTSB medium (Johnson et nl. 1987)

Solution A

Adjust the pH to 2.0 with MSO,. Sterilize by membrane filtration.

Solution B

(NHJ2% 1.26 g/700 mL H,O MgSO, UH,O 0.49 tryp tic soya-broth 0.25

Adjust the pH to 2.5 with H,SO,. Autoclave.

Solution C

agarose 7.0 g/ 250 mL H@

Autoclave. Allow solutions A and B to equilibrate in water bath at 50°C. Allow solution C to cool dotvn in water bath. Add solution A to solution B and mix well. Add slowly the mixture to solution C and mix gently. Pour in plates. Final pH of the medium is 2.5. Table 10. Thiobncillzis medium (ATCC medium 64)

A. Liquid medium

Adjust the pH to 2.8 with H-O,. Sterilize by membrane filtration.

B. Solid medium

Solution A FeSO, 6H,O 10.0 g/350 mL H,O KH,PO, O .? WH,) $0, O -4 MgSO, a7H10 0.08

Adjust the pH to 2.8 with H,SO,. Sterilize by membrane filtration. Allow solutions A to equilibrate inwater bath at 50°C.

Solution B agarose

Autoclave. Allow solution B cool down in water bath for several minutes. Add slowly solution A and mix gentlv. Pour in plates. Table 11. Solidified TK (pH 1.8)

Solution A

Adjust the pH to 1.8 with H,SO,.

Solution B

Adjust the pH to 1.8 using concentrated H,SO,. Mix Solution A and B together. Sterilize by membrane filtration. Allow bo th solutions to equilibrate at 50°C

Solution C

agarose 6 g/300 mL H,O

Autoclave at 121°C for 20 min. Allow solution C to cool down in water bath. Add slowly the mixture to solution C. Mix gently. Pour in plates. APPENDIX 2: SOLUTIONS

Ribonuclease A (bovine pancreas) sterile H,O 1 M ~a~l

Boil for 15 min. Add 0.1 mL of 1 M Tris HC1

Primer extension (PE) rnix

0.9 M WES, 0.1 M MgCl, pH 6.6 1 M Tris-CI pH 7.4 0.1 M dithiothrei tol random hexamers (10 mg/mL) dGTP (50 mM) dATP (50 mM) dTTP (50 mM)

Prehvbridiza tion / hybridiza tion solution deionized H,O 20X SSC 10% (w/v) sodium dodecyl sulfate (SDS) 50X Denhardts denatured salmon sperm DNA (1 mg/mL)

5OX Denhardts solution

Ficoll400 5 g/500 mL of deionized H20 Polyvinylpyrrolidone 5 BSA (bovine serum albumin) fraction V 5

20X SSC

Adjust pH to 7.2 with NaOH. APPENDIX 3: SPECTROPHOTOMETRIC RAW DATA

Table 1. DNA concentration and purity calculated from spechophotometric analysis of standards

standards OD at OD a t OD a t conc (ng/@) 260 /280 320 MI 280 nm 260 nm Table 2. DNA concentration and purity calculated from spectrophotometric analysis of standards included in mixture A and of both mixtures

OD at OD at OD at conc (ng/pL) 260/280 320 nm 280 nrn 260 nm mixture B T.frrrooxidnns 23270 T.ferrooxidnm F1 T.ferrooxidnmi F2 L. jerrooxzdnns CF12 T. aciriophil irs 27807 T.ncidophilrrs 46 T.acidopliiltrs 64 T. tliiooxidmzs 29377 mixture A Table 3. DNA concentration and purity calculated from spectrophotometnc analysis of water samples enriched in different liquid media

- medium OD at OD at OD at conc (ng/pL) 260/280 320 nm 280 nm 260nrn TK 0.0401 0.6492 1.1250 56.250 1.7812 Starkey's Med 1 0.0191 0.5500 0.9393 46.965 1.7331 Tetra thionate I 0.0186 0.8770 1.5988 79.940 1.8409 Tetrathionate2 0.0126 0.1196 0.2008 10.040 1.7393 Glucose 1 0.0086 1.2814 2.2442 112.21 1.7564 Glucose 2 0.0126 0.0935 0.1420 7.100 1.5999 Glucose pH 2.3 0.0084 0.1048 0.1684 8.420 1.6586 Table 4. DNA concentration and purity calculated from spectrophotometric analysis of four thosulfate-oxidizing strains (Mlb, Mld, M2aa, M4b) and one sui fur-oxidizing isola te (B)

strains ODat ODat ODat conc(ng/pL) 260/280 320 nrn 280 nrn 260 nrn Mlb 0.1209 0.6673 1.0561 52.805 1.7115 Mld 0.0442 0.9808 1.6920 84.600 1.7594 Table 5. DNA concentration and purity calculated from spechophotometric analysis of environmental samples from acid mine effluents in Copper Cliff, Ontario

------sample OD at OD at OD at conc (ng/pL) 260/280 320 nm 280 nm 260 nm centrifuged water 0.0352 0.1814 02775 sedimen ts 2.8080 3.1567 3.3420 prefil tered and 0.0760 centrifuged wa ter prefiltered and 3.0840 3.1550 3.2390 fil tered wa ter APPENDIX 4: QUANTITATIVE ANALY SIS

Table 1. Quantitative analysis for cross-hybridization of T. midophilus ATCC 27807

standard PSL PSL-BG conc (ng) k calc [PSL-BG]/c for graph

- kh average 5836.02

PSL: photos tirnulable luminescence BG / bg: background conc/c: concentration of DNA spotted on the filter k calc: calculation of the k,/ k, value A, T. ncidophillrs ATCC 27807; B, T. ncidophiliis 64; C, T.ocidophilzis 46; D, T. thiooxidnns ATCC 19377; E, T. ferrooxidnns ATCC 23270; F, T.firrooxidnm FI; G, T.Jerrooxidms F2; H, L. ferrooxidnns CF12 Table 2. Quantitative analysis for cross-hybridization of T. midophiliis 64

standard FSL PSL-BG conc (ng) k calc [PSL-BG]/c for graph

kA average 5507.50

PSL: photos tirnulable luminescence BG/ bg: background conc/c: concentration of DNA spotted on the filter k calc: calculation of the k,/k, value A, T. acidophiliis ATCC 27807; 8, T. ncidophilirs 64; C, T.ncidophillis 46; D, T. thiooxidlrns ATCC 19377; E, 7'. ferrooxidnns ATCC 23270; F, T. ferrooxidnns FI; G, T. ferrooxidans F2; H, L. ferrooxidnns CF12 Table 3. Quanti ta tive analysis for cross-hybridiza tion of T. aciciophilus 16

------standard PSL PSL-BG conc (ng) k calc [PSL-BG] /c for graph

kh average 4215.03

PSL: photos tirnulable luminescence BG/bg: background conc/c: concentration of DNA spotted on the filter k calc: calculation of the kJk, value A, 7'. nciciophilils ATCC 27807; B, T. ncidophiliis 64; C, T.acidophiluç 46; D, T. thiooxidmts ATCC 19377; E, T.frrrooxidms ATCC 23270; F, T.frrrooxihns FI; G, T.ferrooxidnns F?; H, L. firroowicimis CF12 Table 4. Quantitative analysis for cross-hybridiza tion of T. thiooxidnns ATCC 19377

standard PSL PSL-BG conc (ng) k caic [PSL-BG] /c for graph

kh average 1629.57

PSL: photos timula ble luminescence BG/bg: background conc/c: concentration of DNA spotted on the filter k calc: calculation of the k,/ k, value A, T. ncidophiliis ATCC 27807; B, T. lrcidophilm 64; C, T.lrcidopldzis 46; D, T. thiooxidnns ATCC 19377; E, T. ferrooxidnns ATCC 23270; F, T.ferrooxicinns FI; G, T. ferrooxidnns F2; H, L. ferrooxidnns CF12 Table 5. Quanti ta tive analysis for cross-hybridiza tion of T.fm~ooxidnns ATCC 23270

standard PSL PSL-BG conc (ng) k calc [PSL-BG] /c for graph

kk average 1180.16

PSL: pho tostimulable luminescence BG/ bg: background conc/c: concentration of DNA spotted on the filter k calc: calculation of the kJk, value A, T. ncidophilzis ATCC 27807; B, T. ncidophilris 64; C, T.ncidophiliis 46; D, T. thiooxidnns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T. ferrooxidnns FI; G, T.ferrooxidnns F2; H, L. fmooxidnns CF12 Table 6. Quanti ta tive analysis for cross-hybridiza tion of T.férroowidn~zs Fl

standard PSL PSL-BG conc (ng) k calc [PSL-BG]/c for graph

kh average 1540.84

PSL: photos tirnulable luminescence BG/bg: background conc/c: concentration of DNA spotted on the filter k calc: calculation of the kJ k, value A, T. ncidophiliis ATCC 27807; B, T. ncidophilîrs 64; Cf T.ncidophiliîs 46; D, T. thiooxidnns ATCC 19377; E, T.ferrooxidmzs ATCC 23270; F, T. frrrooxihm FI; G, T.ferrooxidons F2; Hf L. férrooxidnns CF12 Tabie 7. Quanti ta tive analysis for cross-hybridization of T.frrrooxidnns F2

standard PSL PSL-BG conc (ng) k calc [PSL-BG] /c for graph

kh average 1421.17

PSL: photos tirnulable luminescence BG/bg: background conc/c: concentration of DNA spotted on the filter k calc: calculation of the k,/k, value A, T. acidophilris ATCC 27807; B, T. ncidophiiirs 64; C, T.ncidophilils 46; D, T. thiooxidnns ATCC 19377; E, T. ferrooxihns ATCC 23270; F, T. ferrooxidnns FI; G, T.femoxidnns F2; H, L. férrooxidms CF12 Table 8. Quantitative analysis for cross-hybridization of L. ferrooxirinns CF12

standard FÇL PSL-BG conc (ng) k calc [PSL-BG]/c for graph

O

kA average 1734.33

PSL: pho tostimulable luminescence BG/ bg: background conc/c: concentration of DNA spotted on the filter k calc: calculation of the k,/k, value A, T. ncidophiliis ATCC 27807; B, T. ncidophilirs 64; C, T.ncidophilris 46; D, 7'. thiooxidnns ATCC 19377; E, T. ferrooxidnns ATCC 23270; F, T. ferrooxidizns FI; G, T.fmooxidnns F2; H, L. fmooxidnns CF12 Table 9. Quantitative analysis of mixture A of standard genomes

standard PSL PSL-BG conc (ng) khlkx f calc

kA average 17858.80

PSL: photostirnulable luminescence BG/bg: background conc: concentration of DNA spotted on the filter f calc: caldation of the fraction (fJof each standard A, T. flcidophillîs ATCC 27807; 8,T. ncidophilus 64; C,T.ncidophilns 46; Dr T. thiooxidnns ATCC 19377; E, T. ferrooxidans ATCC 23270; Fr T. fmrooxihns FI; G, T.ferrooxidnns F2; H, L. férrooxidnns CF12 Table 10. Quantitative analysis of mixture B of standard genomes

standard PSL PSL-BG conc (ng) kh/kx f calc

kh average 11753.40

PSL: photos tirnulable luminescence BG / bg: background conc: concentration of DNA spotted on the filter f calc: calculation of the fraction (fJof each standard A, T. ncidophiliis ATCC 27807; B, T. ~cidoophilris64; C, T.ncidophilzis 46; D, T. thiooxidnns ATCC 19377; E, T.ferrooxidnris ATCC 23270; F, T. ferrooxidms FI; G, T.ferrooxidnns F2; H, L. ferrooxidnns CF12 Table 11. Quanti ta tive analysis of an environmental sample emiched in TK medium

standard PSL PSL-BG conc (ng) khlkx f calc

kh average 2945.00

PSL: photostirnulable luminescence BG/bg: background conc: concentration of DNA spotted on the filter f calc: calculation of the fraction ( fJof each standard A, T. ncidophiliis ATCC 27807; B, T. ~czdophiiits64; C,T.ncidophiliis 46; D, T. thiooxidnns ATCC 19377; E, T. ferrooxidnns ATCC 23270; F, T. fmooxidnns FI; G, 7'. ferrooxihns F2; H, L. fmuoxidnns CF12 Table 12. Quantitative analysis of an environmental sample emiched in Starkey's medium 1

standard PSL PSL-BG conc (ng) kh/ kx f caic

-- kh average 689.38

PÇL: photostirnulable luminescence BG/bg: background conc: concentration of DNA spotted on the filter f calc: calculation of the fraction (fJof each standard A, T. nciriophil~lsATCC 27807; 8,T. aczdopliil ia 64; C,T.ocidophiliis 46; D, T. thiooxzhns ATCC 19377; E, T.fmooxidnns ATCC 23270; F, T.ferrooxihns FI; G, T.f~rrooxidnns F2; H, L. ferrooxicians CF12 Table 13. Quantitative analysis of an environmental sample emiched in tetrathiona te medium

standard PSL PSL-BG conc (ng) kh/kx f calc

kh average 2730.07

PSL: pho tostirnulable luminescence BG/bg: background conc: concentration of DNA spotted on the fïlter f calc: calculation of the fraction (fJof each standard A, T. ncidophiZils ATCC 27807; B, T. ncidophiliis 64; C, T.acidoophiZus 46; D, T. thiooxicinns ATCC 19377; E, T. ferrooxidnns ATCC 23270; F, T.ferrooxidans FI; G, T.ferrooxidans F2; H, L. ferrooxidnns CF12 Table 14. Quantitative analysis of an environmental sample enriched in glucose medium which was then transferred to tetrathionate medium

standard PSL PSL-BG conc (ng) khlkx f calc

435.20 40 2176.00 kh average 9567.00

PSL: p hotostirnulable luminescence BG/ bg: background conc: concentration of DNA spotted on the fil ter f calc: calculation of the fraction (f,)of each standard A, T. acidophilus ATCC 27807; B, T.ircidophilris 64; C, T.ncidophiliis 46; D, T. thiooxidnns ATCC 19377; E, T. ferrooxidnns ATCC 23270; F, T.ferrooxidnns FI; G, T.ferrooxidirns F2; Hf L. fmoaxid& CF12 Table 15. Quantitative analysis of an environmental sample enriched in glucose medium

standard PSL PSL-BG conc (ng) kh/kx f cak

- kA average 7971.78

PSL: photos tirnulable luminescence BG/bg: background conc: concentration of DNA spotted on the filter f calc: calcula tion of the fraction (fJof each standard A, T. midophilus ATCC 27807; Br T. ncidophilirs 64; C, T.acidophilzis 46; D, T. thiooxidnns ATCC 19377; Er T.ferrooxirlnns ATCC 23270; F, T.jerrooxidnns FI; G, T.ferrooxidms F2; Hf L. ferrooxidnns CF12 Table 16. Quantitative analysis of an environmental sample enriched in a glucose medium (pH 2.3)

standard PSL PSL-BG conc (ng) kh/kx f calc

kh. average 7135.33

PSL: photos tirnulable luminescence BG/ bg: background conc: concentration of DNA spot ted on the fil ter f calc: calc~dationof the fraction (fJof each standard A, T.ncidophi2i.l~ ATCC 27807; Br T. ncidophilris 64; CrT.izcidophiZus 46; D, T. thiooxidnns ATCC 19377; Er T.ferrooxidnns ATCC 23270; F, T. ferrooxidms FI; GrT. ferrooxidlins F2; H, L. ferrooxidnns CF12 Table 17. Quanti ta tive analysis of a hosulfate-oxidizing strain Mlb

standard PSL PSL-BG conc (ng) kh/kx f calc

kh average 3053.75

PSL: photos timulable luminescence BG/ bg: background conc: concentration of DNA spotted on the filter f calc: calculalion of the fraction (fx)ofeach standard A, T. miciophilits ATCC 27807; 8, T. ncidophilrls 64; C, 7'.nci(i0phillis46; D, T. thiooxicinns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T. ferrooxidnns FI; G, T.ferrooxidms F2; H, L. fermoxidnns CF12 Table 18. Quanti ta tive analysis of a thiosulfa teoxidizing strain Mld

standard PSL PSL-BG conc (ng) f calc

kh average 3575.00

PSL: photos tirnulable luminescence BG/ bg: background conc: concentration of DNA spotted on the filter f calc: caldation of the fraction (f,)of each s hndard A, T. ncidophilils ATCC 27807; B, T. ncidophiliis 64; Cf T.ncidophillis 46; D, T. thiooxidnris ATCC 19377; E, T. ferrooxidnns ATCC 23270; F, T. ferrooxidnns FI; G, T.fenaoxidnns F2; HfL. fmooxidnns CF12 Table 19. Quanti ta tive analysis of a thiosdfa te-oxidizing strain Waa

standard PSL PSL-BG conc (ng) kh/kx f calc

kh average 6949.17

PSL: photos tirnulable luminescence BG/ bg: background conc: concentration of DNA spotted on the filter f calc: calculation of the fraction (f,)of each standard A, 7'. ~~id~phdlt~ATCC 27807; 0, T. ncidophilus 61;C, T.ncidophiliis 46; D, T. thiooxicinns ATCC 19377; E, T.ferrooxihns ATCC 23270; F, T.frrrooxidnnç FI; G, T.ferrooxidnns F2; Hf L. ferrooxidnns CF12 Table 20. Quantitative analysis of a thiosulfate-oxidizing strain M4b

- .- . standard PSL PSL-BG conc (ng) u/kx f calc

ld average 768.16

PSL: photos tirnulable luminescence BG/bg: background conc: concentration of DNA spotted on the filter f calc: calculation of the haction (fJof each standard A, T. ncidophiliis ATCC 27807; £3, T. ~cidophilr~s64; C, T.ncidophilus 46; D, T. thiooxidnns ATCC 19377; E, T.jerrooxidnns ATCC 23270; F, T. ferrooxidnns FI; G, T.ferrooxidnns F2; H, L. ferrooxidnns CF12 Table 21. Quantitative analysis of a sulfur-oxidizing culture B (analysis 1)

standard PSL PSL-BG conc (ng) khlkx f calc

kh average 663.53

PSL: photos tirnulable luminescence BG / bg: background conc: concentration of DNA spotted on the filter f calc: calculation of the fraction (f,)of each standard A, T. ncidophihis ATCC 27807; B, T. ncidophilils 64; C, Tmidophiliis 46; D, T. thiooxirinns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T. ferrooxidnns FI; G, T.fenooxidnns F2; H, L.ferrooxidnns CF12 Table 22. Quantitative analysis of a sulfur-oxidizing culture B (analysis 2)

standard PSL PSL-BG conc (ng) khlkx f calc

kh average 745.47

PSL: photos tirnulable luminescence BG/ bg: background conc: concentration of DNA spotted on the filter f calc: calculation of the fraction (fJof each standard A, T. ncidophillrs ATCC 27807; B, T. ~czdophihs64; C, T.ncidophilus 46; D, T. thioosidnns ATCC 19377; E, T. ferrooxidnns ATCC 23270; F, T. ferrooxidms FI; G, T.ferrooxidans F2; H, L. ferrooxidnns CF12 Table 23. Quantitative analysis of an environmental water sample centnfuged to collect cells (analysis 1)

standard PSL PSL-BG conc (ng) khlkx f calc

kh average 1612.08

PSL: photos tirnulable luminescence BG/ bg: background conc: concentration of DNA spotted on the filter f calc: calcula tion of the fraction (f,)of each standard A, T. ncidophilzis ATCC 27807; B, T. ciciciophil~is64; C,T.acidophihs 46; D,T. tliiooxidnns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T.ferrooxidans FI; G,7'. ferrooxidnns F2; H,L. ferrooxidnns CF12 Table 24. Quantitative analysis of an environmental water sample centrifuged to collect cells (analysis 2)

standard PSL PSL-BG conc (ng) khlkx f calc

kh average 1539.79

PSL: photos timulable luminescence BG/bg: background conc: concentration of DNA spotted on the filter f calc: calculation of the fraction (f,)of each standard A, T. ncidophilris ATCC 27807; B, T. ~cidophillrs64; C, T.~cidophilris46; D, T. thiooxidnns ATCC 19377; E, 7'. ferromirinns ATCC 23270; F, T.fërrooxidnns FI; G, T.ferrooxidnns F2; H, L. ferrooxidnns CF12 Table 25. Quantitative analysis of an environmenta1 wa ter sample prefiltered and centrifuged to collect cells

standard PSL PSL-BG conc (ng) kh/kx f calc

O O 10 265.04 20 220.83

40 121-04 kh average 202.31

PSL: pho tostimulable luminescence BG/bg: background conc: concentration of DNA spotted on the filter f calc: calculation of the fraction (fJof each standard A, T. cid do ph il ils ATCC 27807; B, T. cid do ph il ils 64; C, T.ncidophiltls 46; D, T. thiooxidnns ATCC 19377;E, T.ferrooxidnns ATCC 23270; F, T.ferrooxidnns FI; G,T. ferrooxidnns F2; H,L. ferrooxidmis CF12 Table 26. Quantitative analysis of an environmental sediment sample

ç tandard PSL PSL-BG conc (ng) kh/kx f calc

O O 10 1616.88 20 1389.58 40 867.19 kh average 1291.22

PSL: photostirnulable luminescence BG/ bg: background conc: concentration of DNA spotted on the filter f calc: caldation of the haction (fJof each standard A, T.ncidophilils ATCC 27807; B, T. ncidophilus 64; C, T.ncidophi1ii.s 46; D, T.thiooxidnns ATCC 19377; E, T.ferrooxidnns ATCC 23270; F, T. furooxidnizs FI; G, T.fewooxidnns F2; H, L. ferrooxidnns CF12