Isolation and Characterization of Iron-Oxidizing Bacteria

Home , Lipophilic bacteria, Pyrolobus fumarii, Sulfobacillus

ISOLATION AND CHARACTERIZATION OF IRON-OXIDIZING BACTERIA

FROM BOILING SPRINGS LAKE AND THE POTENTIAL ROLE OF FERROUS

IRON IN CARBON AND SULFUR CYCLING

Francine Arroyo

A Thesis

Presented to

The Faculty of Humboldt State University

In Partial Fulfillment of the Requirements for the Degree

Master of Science in Biology

Committee Membership

Patricia L. Siering, Ph.D., Committee Chair

Mark S. Wilson, Ph.D.

Matthew Hurst, Ph.D.

Kristine Brenneman, Ph.D.

Michael Mesler, Ph.D., Graduate Coordinator

December, 2012 ABSTRACT

ISOLATION AND CHARACTERIZATION OF IRON-OXIDIZING BACTERIA

FROM BOILING SPRINGS LAKE AND THE POTENTIAL ROLE OF FERROUS

IRON IN CARBON AND SULFUR CYCLING

Francine Arroyo

Boiling Springs Lake (BSL) is a 52˚C, pH 2, iron and sulfur-rich thermal feature

in Lassen National Volcanic Park (California, USA). Previous community composition

studies of small subunit rRNA and RuBisCo genes revealed an abundance of phylotypes closely related to an Acidimicrobium strain isolated from Yellowstone National Park. As an ideal candidate to examine its contribution to primary production in BSL, we attempted to isolate Acidimicrobium and related iron-oxidizing Bacteria from BSL. We obtained 23 isolates that shared 99% rRNA gene identity with their closest cultured relative: 16 were identified as Sulfobacillus acidophilus, four as Alicyclobacillus sp., and three isolates had nearly identical rRNA gene sequences to the previously identified

Acidimicrobium clones. We characterized most isolates for pH and temperature growth range and optima, and we assessed their abilities to oxidize iron, pyrite, sulfur and tetrathionate. Morphology of Acidimicrobium isolates was analyzed with transmission and scanning electron microscopy. The Acidimicrobium isolates were Gram positive, non-endospore-forming rods with a complex cellular envelope. Optimal growth ii temperature and pH for many isolates correlated with conditions at BSL. Acidimicrobium and Sulfobacillus isolates oxidized 10 mM iron when amended with 0.01% yeast extract.

Acidimicrobium isolates were inhibited at iron concentrations ≥25 mM. Acidimicrobium and some Sulfobacillus isolates were able to oxidize pyrite when amended with yeast extract. We found no evidence for sulfur or tetrathionate oxidation by any of the isolates.

These results will further our understanding of the potential role of Acidimicrobium and related iron-oxidizing Bacteria in the iron and carbon cycles in BSL.

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ACKNOWLEDGEMENTS

I would first like to thank my advisor, Patricia Siering, for her unyielding support

and time dedicated to making sure that I complete my thesis in a timely manner. I cannot

accurately express my gratitude for her efforts these past three and a half years. She is a

role model for hard work, passion for teaching, and all-around joyful person to work

with. I can only hope to maintain my head above water in the wake that she leaves

behind.

Many thanks to my committee members for their continued support and time

dedicated for reviewing this endless thesis. Mark was always available to answer any

questions that I would have, from assistance with sequence alignments to recipes for

posole soup. Many thanks to Matt for all his help in setting up the iron oxidation assays and allowing me access to the Chemistry department’s diode array spectrophotometer.

To Dr. B for showing me how much fun it is to monitor the Secchi depth.

My work utilizing electron microscopy could not have been done without the patience and guidance of Casey Lu. I would also like to thank Marty Reed and Lewis

McCrigler for helping me through the technical difficulties with the microscopes.

Dr. Barrie Johnson from the University of Wales (Bangor, U.K.) provided me with insight on successful growth maintenance of the finicky A. ferrooxidans ICPT.

Many members of the H.E.A.T. lab contributed to my work. Jennifer Hampton’s

initial attempts of isolating Acidimicrobium provided me with the framework of methods

and media to use in my isolation attempts. Jon Schultz and Clay Carey assisted me with iv

clone library construction. Connor Fitzhugh eagerly helped me analyze my experimental

data using AICc under the guidance of Dr. Rob Van Kirk.

I would like to thank my partner, Max Cannon, for moving to Arcata with me and personally supporting me through my emotional vicissitudes.

Thanks to the helpful and optimistic staff and faculty of the Department of

Biology. It has been a pleasure.

Funding was provided by the National Science Foundation (Patricia Siering, PI,

grant no. MCB-0702018).

TABLE OF CONTENTS

ABSTRACT ...... ………..ii

ACKNOWLEDGEMENTS ...... ………iv

TABLE OF CONTENTS……………………………………………………………..…..vi

LIST OF TABLES…………………………………………………………………….…..x

LIST OF FIGURES…………………………………………………………………....…xi

LIST OF APPENDICES………………………………………………………...……...xiii

CHAPTER 1: INTRODUCTION ...... 1

Project Overview……………………………………………………………….....1

Literature Review: Physiology of Acidophiles……………………………………2

Coping mechanisms for an acidophilic lifestyle…………………………..2

Thermoacidophily…………………………...…………………………….4

Metabolic diversity of acidophiles………………………………………...7

Iron and sulfur oxidation…………………………………………….…….9

Background on Boiling Springs Lake…………………...……………………….13

Site description……………………………………………………..….…13

Microbiology of BSL………………………………………………….…16

Production in BSL……………………………………………………….17

Comparison with acid mine drainage sites………………………………18

CHAPTER 2: MATERIALS AND METHODS ...... 19

Media Preparation………………………………………………………….…….19

Isolation of Sulfobacillus and Alicyclobacillus Strains…………………….…….22

Sample collection……………………………..………………………….22

Winogradsky column enrichments…………………………………...….23

Selection and purification of isolates…………………………………….24

Isolation of Acidimicrobium sp. from BSL……………….……………….……..26

Sample collection………………………………………………….……..26

Isolation and purification…………………………………………...……26

Identification of Isolates by 16S rRNA Gene Sequencing………………………29

Acidimicrobium-specific 16S rRNA primer design……………………...29

SSU rRNA gene amplification by PCR………………………………….30

Clone library construction and sequence analysis……………………….31

Morphology of Acidimicrobium by Electron Microscopy…………………….…34

Transmission electron microscopy (TEM)……………………………....34

Scanning electron microscopy (SEM)……………………………...……35

Physiological Characterization of Isolates……………………………………….35

Preparation of inocula and growth assessment…………………………..35

Determination of pH and temperature range and optima for growth…….37

Growth experiments………………………………………...……37

Linear regression models and Akaike information criterion

(AICc)……………………………………………………….……37

Iron and pyrite as energy sources…………………………………….…..38

Iron and pyrite oxidation via ferrozine assay…………………………….39 vii

Sulfur and tetrathionate oxidation………………………………………..40

CHAPTER 3: RESULTS ...... 41

Isolation, Identification and Naming of Alicyclobacillus and Sulfobacillus

Isolates…………………………………………………………………………...41

Isolation, Identification and Naming of Acidimicrobium Isolates…...…………..43

Electron Microscopy of Acidimicrobium…………………………………...……50

Physiological Characterization of Isolates……………………………………….54

Determination of temperature and pH range and optima………….……..54

Growth experiments……………………………………….…..…54

Linear regression modeling of pH and temperature data………...64

Evaluation of iron and pyrite as potential energy sources……………….69

Measurements of iron oxidation……………………………………...….74

Pyrite oxidation…………………………………………………………..75

Sulfur and tetrathionate oxidation………………………………………..80

CHAPTER 4: DISCUSSION ...... 86

Attempts to Isolate Acidimicrobium from BSL……………………………….…86

Isolation and physiological characterization of Acidimicrobium…....…..89

Cell morphology of Acidimicrobium…………………………………….97

Caveats associated with growth measurements…………………..…….100

Isolation and physiological characterization of Sulfobacillus…..…..….100

Using AIC analysis to differentiate between strains of Sulfobacillus

and between species of Sulfobacillus and Acidimicrobium…….…....…103 viii

Isolation and physiological characterization of Alicyclobacillus….……103

Speculation on Role of Acidophilic Isolates in BSL…………….…………..…106

Summary………………………………………………………………..………109

Future Work…………………………………………………………………….111

APPENDICES…………………………………………………………………….……115

REFERENCES ...... 119

LIST OF TABLES

Table Page

1 16S rRNA primers utilized……………..……………………………………..…33

2 16S rRNA gene sequence analysis, isolation conditions, and morphology of isolates…………………………………………………………………..…….…46

3 Summary of temperature and pH ranges and optima for Acidimicrobium, Sulfobacillus, and Alicyclobacillus isolates………………………….……..……63

4 AICc of the top linear regression models of division rate (generations/time) according to pH, temperature, and strain of Sulfobacillus isolates.……….……..66

5 Sulfobacillus isolate sub-groups organized by common temperature and pH ranges/optima………………………………………………………………….....67

6 AICc of the top linear regression models of division rate (generations/time) according to pH, temperature, strain, and/or sub-groups of Sulfobacillus sp. isolates...... 67

7 AICc of the top linear regression models of division rate (generations/time) according to pH, temperature, and species of Acidimicrobium and Sulfobacillus…………………………………………………………………….. 68

8 Summary of iron, pyrite, sulfur and tetrathionate oxidation results for Acidimicrobium and Sulfobacillus……………………...……………………..…85

LIST OF FIGURES

Figure Page

1 Model of electron transport chain in Gram negative Acidithiobacillus ferrooxidans……………………………………………………………………..12

2 Boiling Springs Lake (BSL) with sites A and D designated by arrows…….…...15

3 Flow chart schematic summarizing methods for isolation of Sulfobacillus and Alicyclobacillus strains………………………………………………………..…25

4 Flow chart schematic summarizing methods for isolation of Acidimicrobium sp………………………………………………………………………………....28

5 Comparison of colony morphology between Sulfobacillus and Acidimicrobium isolates……………………………………………………………………………48

6 Gram stained photographs of representative Sulfobacillus isolates (a), Alicyclobacillus isolates (b), and Acidimicrobium isolates (c) and A. ferrooxidans ICPT (d) viewed by 1000X light microscopy…………………………………….49

7 Electron micrographs of Acidimicrobium EAO4 in Heterotrophic medium….…52

8 Cell morphology comparison of Acidimicrobium EAO2 (left) and A. ferrooxidans ICPT (right) in Mixotrophic medium………………………………………..…...53

9 Three patterns of temperature growth for Sulfobacillus isolates represented by JAO2 (a), JWO20m (b), and JWO22m (c)………………………………………...57

10 Three patterns of pH ranges and optima for Sulfobacillus isolates…………..….58

11 Three patterns for permissible temperature range of growth for Alicyclobacillus isolates……………………………………………………………………….…..59

12 Unique patterns for pH range and optima for Alicyclobacillus isolates…..……..60

13 Comparison of temperature range and optima between Acidimicrobium isolate EAO2 and A. ferrooxidans ICPT…………………………………………...…….61

LIST OF FIGURES (CONTINUED)

Figure Page

14 Comparison of pH range and optima between Acidimicrobium isolate EAO2 and A. ferrooxidans ICPT…………………………………………………………..…62

15 Comparison of autotrophic growth at varying iron concentrations, supplemented T with either air or enhanced CO2, between A. ferrooxidans ICP (top) and isolate EAO4 (bottom)……………………………………………………………...…...72

16 Mixotrophic growth comparison of A. ferrooxidans ICPT (a), Acidimicrobium EAO1 (b), and Sulfobacillus JWO19m (c)………...………………………..……73

17 Comparison of growth and concomitant iron oxidation by Acidimicrobium and Sulfobacillus strains………………………………………………………..….…77

18 Comparison of iron oxidation in 1% Pyrite medium by Acidimicrobium EAO4 (a) and Sulfobacillus JWO19m (b)…..……………………………..………….…78

19 Comparison of dissolution of pyrite by Acidimicrobium (a) and Sulfobacillus (b) isolates………………………………………………………………………...….79

20 Comparison of growth in Sulfur and Tetrathionate media by A. ferrooxidans ICPT (a) and Acidimicrobium isolate EAO4 (b)……………………………………….83

21 Comparison of growth in Sulfur and Tetrathionate media by Sulfobacillus isolates JWO19m (a) and JWO19m (b)………………………………………………..….84

xii

LIST OFAPPENDICES

Appendix Page

A Freezing of cultures……………………………………………………...... 115

B Maintenance of cultures……………………………...…………………………116

C Statistical equations used for linear regression…………………………………117

xiii

CHAPTER 1: INTRODUCTION

Project Overview

Boiling Springs Lake (BSL) is a 50-93˚C, pH 2, oligotrophic iron and sulfur-rich

thermal feature located in Lassen National Volcanic Park (California, USA). The

compounded stresses of high temperature, low nutrients and pH select for a microbial

community capable of tolerating and thriving at the extremes of life. The abundance of

reduced iron (102, 103) and presumably sulfur species in BSL provides possible energy-

generating mechanisms for organisms living in the hot spring. Sequences from small

subunit rRNA genes revealed representatives from each of the domains of life, wherein

novel Archaea and Bacteria appear to dominate the sediment community, and Bacteria closely related to Acidimicrobium and Hydrogenobaculum spp. appear to be the most abundant organisms in the water column (116, 117). The aim of this study was to investigate the role of an abundant Bacteria phylotype (Acidimicrobium spp.) detected in

SSU rRNA and RuBisCo gene clone libraries (102, 117). Our goals were to (i) isolate

Acidimicrobium spp. and/or physiologically/phylogenetically similar bacteria, (ii) investigate morphological characteristics of Acidimicrobium utilizing electron microscopy, and (iii) determine optimal growth conditions of isolates and their ability to oxidize iron, sulfur, and pyrite. By studying the autecology of thermoacidophilic, iron- oxidizing Acidimicrobium and other members of the community, we can make better predictions of ecological interactions and active biogeochemical processes in BSL.

Literature Review: Physiology of Acidophiles

Coping mechanisms for an acidic lifestyle

Generally, there are two categories of acidophiles. Extreme acidophiles have pH optima for growth at <3, and moderate acidophiles grow optimally at pH 3-5 (52). Acid- tolerant organisms that are metabolically active at low pH, have a growth optima of pH

≥5. In this study, we will focus on the physiology of extreme acidophiles, herein referred to as acidophiles. Although the environment in which acidophiles live is low in pH, acidophiles require an intracellular pH close to neutral (82). In order to maintain a pH

homeostasis in contrast to an external environment with a concentration of protons 10-4 M

or higher, acidophiles have adapted many coping mechanisms to survive and thrive in

acidic environments.

Acidophiles have a highly impermeable membrane that prevents ingress of protons (10). Surprisingly, no differences in the structure of cell membranes between acidophilic and neutrophilic bacteria have been identified (54). Yet, many Archaea, such as Ferroplasma acidiphillum YT, Thermoplasma acidophilum, Sulfolobus solfactaricus, and Picrophilus oshimae have membranes composed of tetraether lipids (41, 95, 101,

112). Ether linkages are less sensitive to acid hydrolysis than bacterial (and eukaryal) ester linkages (54).

Similar to neutrophiles, acidophiles also utilize pH gradients and membrane potentials to generate a proton motive force (PMF) (10). PMF is essential for converting

ATP from ADP using membrane-bound ATPases, transporting substances across

membranes, and driving flagella movement in flagellated Bacteria (54). In contrast to

neutrophiles, acidophilic bacteria actively pump protons out of the cell often in concert with symport or antiport of other substances to maintain homeostasis at a cost of ATP.

Acidophiles also actively pump cations such as K+ into the cell, resulting in a more positive intra-membrane potential than neutrophilic cells (1). The positive internal

membrane potential protects acidophilic cells when ATP is limiting, thereby preventing

acidification of the cytoplasm which leads to death (71). The positive membrane potential

also confers some protection against many positive charged cations, such as transition

metals that are often present at high concentrations in acidic environments.

Consequences of possessing a positive internal membrane potential include

increased sensitivities to both low molecular weight organic acids (i.e. acetic and pyruvic

acids) and common anions like nitrate (82). Due to the pKa values for many of these

organic acids in acidic environments, the acids occur in the protonated form and can be

transported into the cells of acidophiles. Protonated acids dissociate in the circumneutral

cytoplasm and result in acidification of the cytoplasm. Interestingly, all acidophiles able

to grow at extremely acidic pH (<0) are heterotrophs that have genes encoding organic

acid degradation pathways (6). However, it is not known whether the ability to degrade

organic acids is a pH homeostatic mechanism.

Proteins and enzymes located on the exterior of the cell have adapted to be acid-

stable and active at low pH. Generally, proteins that are dominated by α-helical structures

have a reduced charge density (72). As pH decreases, protonation of acidic residues

increases, leading to protein unfolding due to the increase in repelling forces. Therefore, a

4 reduced number of acidic residues and negative charges result in increased acid stability.

Proteins dominated by β-sheet structures, such as the electron carrier protein rusticyanin, are inherently more acid-stable and do not require reduced charge densities (113). Due to the high redox potential of rusticyanin at low pH (Eh=0.68V), it is optimally active at pH values ≤2.

Lastly, acidophiles have a large number of DNA and protein repair genes that are necessary when pH homeostasis is not maintained (31, 51, 88). In a proteome expression study of an AMD biofilm community containing Leptospirillum group II, chaperones involved in protein refolding were highly expressed (11% of total proteome) (88).

Chaperonins in Acidothiobacillus ferrooxidans were also found to be upregulated when the acidophile experienced a drop in medium pH from 3.5 to 1.5 (51). However, upregulation of chaperonins was not observed when the medium pH was increased from

1.5 to 3.5.

Thermoacidophily

Acidophiles with optimum pH<3 may be categorized into three groups by their response to temperature: mesophilic acidophiles (optima < 40˚C), moderate thermoacidophiles (optima of 40-60˚C), and extreme thermoacidophiles (optima >60˚C)

(52). These three temperature categories also correlate with taxonomy; the majority of mesophilic acidophiles are Gram negative bacteria in the Proteobacteria phylum, most moderate thermo-acidophiles are Gram positives in the Firmicutes and Actinobacteria phyla, and all known extreme thermo-acidophiles are Archaea, with the notable

exception of Hydrogenobaculum (in the Aquificales phylum in Bacteria). The highest known temperature limits for growth at circumneutral pH by phototrophy, chemoheterotrophy, and chemoautolithotrophy, are 75°C (3), 113˚C (13), and 122˚C

(108), respectively. The record for the highest temperature of growth is held by

methanogenic archaeon Methanopyrus kandleri at 122˚C. Based on the upper limits of

temperature stability for ATP, amino acids and peptides, the maximum temperature limit for life is estimated at ~150°C (114, 115). However, since many of the structural and physiological stresses associated with living in acidic habitats are similar to (and compounded by) also living at high temperature - it is unlikely that thermoacidophily would function at such a high temperature. For instance, the most acidophilic prokaryote known is archaeon Picrophilus oshimae that grows optimally at pH 0.7 and grows at temperatures between 45-65˚C (opt. 60˚C) (6, 98). Acidianus infernus is able to grow at the highest temperature (range 65-96˚C; optimum 90˚C) and lowest pH (1-5.5; optimum

~2) combination known to support growth (99).

Cells must be able to maintain their PMF in order to sustain growth at high temperatures. Ion permeability increases with increasing temperatures, which can disrupt the PMF (112). However, the acidophile’s positive internal membrane potential helps alleviate some of the stresses from increased temperatures by preventing ingress of protons. In addition, cell membranes of Bacteria living in high temperature environments are adapted to limit proton permeation by increasing the degree of saturated fatty acids, increasing chain length of acyl chains, and increasing chain branching (86, 90, 107). As a result, these heat-adapted membranes are able to maintain a liquid-crystalline structure in

spite of the increased entropy induced by high temperatures. As mentioned earlier, many

Archaea contain glycerol tetraether lipids in their membranes which form covalently

linked mono-layers. These mono-layer lipids increase the stability of the membrane and

reduce proton permeation (54).

Unlike acid-stable proteins, thermal stability of proteins tend toward a higher abundance of charged amino acid residues with increasing growth temperature, and reduced frequency of polar residues (6, 60, 96). In theory, electrostatic interactions,

including salt bridges, increase internal bonding of proteins due to their long range (4Å)

in comparison to van der Waals forces. An increase in protein surface charge increases

intracellular solubility of proteins (20).

Extreme thermophiles contain unique chaperonins to prevent and protect against

protein denaturation. Heat shock proteins (HSP) have been correlated with adaptive

thermotolerance across all domains of life (94). The first evidence for the role of HSP in

thermotolerance was found in the hyperthermophilic archaeon Sulfolobus shibatae (110,

111). When S. shibatae was exposed to 88°C for 60 minutes, cells were able to thrive for

40 minutes at a normally lethal temperature of 95°C. High levels of chaperone expression

for hsp60 accompanied the acquired heat tolerance.

There are no recognizable differences in genome sequence content as a whole

between thermophiles and mesophiles, however there is a bias for increased GC content

in tRNA and rRNA of thermophiles (49). The additional hydrogen bond between GC

base pairs (versus AU base pairs) provides a more thermally resistant double stranded

RNA molecule. Coding sequences of mRNAs of thermophiles also show a preference for

certain dinucleotides (ex. CC and GG) at the first and second position of codons (60).

Secondary structures of nucleic acids are stabilized by monovalent and divalent

cations (Na+, K+, and Mg2+), as well as linear and branched polyamines (70). Cations and

polyamines shield the highly negative phosphate backbone of DNA and RNA to facilitate

proper folding. Moreover, hyperthermophiles contain a family of DNA topoisomerases called reverse gyrases that produces negative supercoiling (58). Generation of negatively

supercoiled DNA provides additional thermal protection to dsDNA that is positively

supercoiled in most mesophilic organisms.

Metabolic diversity of acidophiles

Chemolithotrophy is more prevalent among acidophiles than it is in any other physiologic group of microorganisms (52). Chemolithothrophy is the ability to derive energy from purely inorganic sources. Chemolithotrophs are often numerous in low pH environments because inorganic energy sources are usually more abundant than organic carbon (53). The extremely acidic (pH 0.8 to 1.38) and metal-rich waters in the

Richmond mine at Iron Mountain are dominated by iron-oxidizing bacteria Ferroplasma sp. and Leptospirillium spp. (34). Leptospirillium ferrooxidans and iron- and sulfur-

oxidizing Acidithiobacillus ferrooxidans are also the most commonly detected Bacteria

in the acidic (~pH 2) cool waters (15-25°C) of Tinto River in southwestern Spain (42). In

addition, chemolithotrophy is considered the source of primary production in

environments lacking sunlight like deep sea vents (78) and in environments too hot for

photoautotrophic growth such as thermal hot springs (12).

Mixotrophs may derive energy from inorganic compounds while requiring organic compounds for their carbon needs. Simultaneous assimilation of an inorganic energy source and organic carbon provides mixotrophs with an advantage in acidic environments that have fluctuating concentrations of organic carbon or reduced inorganic energy sources (43). Heterotrophic acidophiles utilize organic compounds as both carbon and energy sources and can grow aerobically, anaerobically, or both, depending on the species and environmental conditions.

The redox potential of environments that prokaryotes inhabit can vary. When present, oxygen is the most commonly used electron acceptor (esp. for iron oxidizers) due to the high redox potential of +1.2 mV (at pH 2). However, most environments that acidophiles and/or thermophiles occupy are anoxic or reducing in nature because of the inputs of gases like H2 and H2S and reduced oxygen solubility at elevated temperatures

(18, 106). Alternative electron acceptors for anaerobic respiration include ferric iron and sulfate. At pH<2.5, the ferrous/ferric couple reaction has a strong positive redox potential

(+0.77 V) that is slightly less electropositive than the oxygen/water couple. Therefore ferric iron is an attractive alternative terminal electron acceptor to molecular oxygen.

Although the theoretic energy yield is reduced when growing anaerobically versus aerobically, there is an advantage for anaerobic autotrophic growth. A study calculating energy requirements for biomass synthesis by chemolithoautotrophs between oxic and anoxic environments found a 10-fold greater energy requirement for aerobes relative to anaerobes (74).

Iron and sulfur oxidation

Iron sulfides, notably pyrite (FeS2), are the most abundant sulfide minerals in the lithosphere and constitute an important reservoir of reduced sulfur and iron. Ferrous iron

(Fe2+) is highly soluble and readily auto-oxidized to insoluble ferric iron (Fe3+) by

molecular oxygen at pH values greater than 5 (36). At low pH (<5), ferrous iron is less

susceptible to auto-oxidation. Therefore, most evidence for the enzymatic oxidation of iron is from studies at acidic pH (36). Thermodynamically, oxidation of ferrous iron is not a lucrative energy source (6.5 kcal/mol at pH 2.5) since it barely produces enough energy for the synthesis of 1 mol of ATP (7 kcal/mol) (64). In order to provide enough energy for autotrophic metabolism, approximately 18.5 mol of Fe2+ would have to be

oxidized to fix 1 mol carbon (assuming 120 kcal to produce 1 mol C at 100% efficiency)

(105). CO2 fixation by the Calvin Benson pathway requires NAD(P)H which is produced

by the reduction of NAD(P)+ by electrons obtained during growth. Studies of

Acidithiobacillus ferrooxidans (87) describe this process utilizing Fe2+ as the reducing

power. The redox potential of iron oxidation is more positive than the

NAD(P)+/NAD(P)H couple, therefore electrons must be transported ‘uphill” against the

electropotential gradient (Fig. 1). This process consumes energy in the form of ATP and is termed reverse electron transport. Although the free energy associated with the oxidation of ferrous iron is very low, the high concentration of ferrous iron in many acidic habitats allows it to be an important electron donor for many acidophiles.

Ferric iron is also very soluble and abundant at pH<2.5 (36). In addition to the use of Fe3+ as an alternative electron acceptor, ferric iron has been argued to be the major

oxidant of pyrite (38, 97) as:

3+ 2+ 2- 2+ + FeS2 + 6Fe + 3H2O Fe + S2O3 + 7Fe + 6H .

2- At low pH, thiosulfate (S2O3 ) is not stable and hydrolyzes to various forms of

2- polysulfides, as well as sulfate (SO4 ) and elemental sulfur. Currently, it is impossible to

distinguish between direct and indirect oxidation of pyrite by microbes. However, due to

3+ the preference of Fe over O2 as the chemical oxidant of pyrite, iron-oxidizing bacteria can facilitate the oxidative dissolution of pyrite in the presence of oxygen.

Sulfur is one of the most abundant elements in the lithosphere and is an essential nutrient for all life forms (52). Although biological processes such as fermentation and nitrification generate acidity, sulfur oxidation processes are responsible for generating

most of the extremely acidic habitats around the world (54). Oxidation of sulfur by

autotrophic and heterotrophic microorganisms generates sulfuric acid as:

S˚ + H2O + 1.5O2 H2SO4.

In the absence of carbonates or other basic minerals or buffers, dramatic decreases

in pH will result from sulfur oxidation. Some chemolithotrophic prokaryotes can obtain

some or all of their energy needs from the oxidation of elemental sulfur (S˚) and/or

various reduced inorganic sulfur compounds (RISCs). Utilization of S˚ and RISCs are

more energetically favorable than ferrous iron (52). Elemental sulfur and RISCs (such as

thiosulfate and tetrathionate) provide more electrons per mole of sulfur than ferrous iron.

For example, one electron from the oxidation of Fe2+ to Fe3+ is released, versus six

2- electrons from the oxidation of S˚ to SO4 . Also, the electrons obtained from the

oxidation of S˚ and RISCs enter the electron transport chain at a higher redox potential

than electrons from ferrous iron; therefore, more ATP is produced per mole of substrate.

2- For example, the free energy available (at pH 2) from the oxidation of S2O3 to H2SO4 is

2+ -762.47kJ/mol while Fe oxidation is -138.89 kJ/mol. During CO2 fixation by

autotrophs, electrons are thus readily available for the reduction of NAD(P)+ to

NAD(P)H, eliminating the need for reverse electron transport. This effect is evident in

the disproportionately higher growth yields obtained when Acidothiobacillus

ferrooxidans is grown on sulfur versus iron (45) and the greater cell densities of autotrophic sulfur oxidizers over iron oxidizers in bioreactors (84).

Fe2+ Fe3+

OM Cyc2

Rus PERIPLASM

Cyc c4 Cyc c4

Cytochrome NADH IM bc1complex UQ oxidase dehydrogenase

+ + 2 H + ½ O2 H2O NAD(P) + H NAD(P)H CYTOPLASM

Figure 1. Model of electron transport chain in Gram-negative Acidithiobacillus ferrooxidans (87). During the oxidation of ferrous iron to ferric iron, electrons are used to reduce oxygen and protons to water (solid straight arrows). However, when utilizing electrons from ferrous iron for CO2 fixation, electrons are transported “uphill” (dotted straight arrows) by the electron transport chain to NAD(P) via ubiquinone (UQ). Key: OM, outer membrane; IM, inner membrane; RUS, rusticyanin; Cyc, cytochrome c.

Background on Boiling Springs Lake

Site description

Lassen Volcanic National Park (LVNP) is located at the south end of the Cascade

Range and features an array of hydrothermal activity, including the large, hot, acidic lake

known as Boiling Springs Lake (BSL). BSL is the largest hot spring in North America

with a surface area of ~18,000m2; the size and pH appear relatively stable from direct

summer measurements (1999-2012) despite seasonal variations (27, 102, 103). BSL is a

designated National Science Foundation Microbial Observatory, in which the ecosystem

within this hydrothermal lake is being investigated in our laboratory in collaboration with

biologists Gordon Wolfe at Chico State University and Kenneth Stedman at Portland

State University.

The LVNP hydrothermal features result from a vapor-dominated system that

produces acidic waters (~pH 2.0). BSL has lower chloride (~1 ppm) and higher sulfate

(~967 ppm) (102, 103) concentrations than most of the liquid-dominated geothermal

systems in Yellowstone National Park (50). Compared to other acidic systems (34, 42,

57), heavy metals in BSL (other than Fe; ~38 ppm) are present in low amounts (<0.1

ppm) (102, 103). BSL is oligotrophic (TOC and DOC ~1 ppm) despite receiving major

inputs of allochthonous organic material from surrounding alpine forest (102). The

geothermal hot spring is a well-mixed water feature, filled with suspended sediments of mostly fine clay that result in a secchi depth of <10 cm (102, 117). The majority of BSL

and site A (Fig. 2) have a summer temperature range of 50-55°C (102, 103, 116) while

winter temperatures range from 39.8-48˚C (39, 117). Active geothermal inputs can be observed in the south end of the lake (Fig. 2, site D) with summer temperatures ranging from 65-95°C (103, 116, 117).

Figure 2. Boiling Springs Lake (BSL) with sites A and D designated by arrows. BSL is an acidic (pH ~2), thermal, iron-sulfide-rich hot spring in Lassen National Volcanic Park, CA, USA. Average summer water temperature of sites A and D are 52˚C and 82˚C, respectively. Average winter water temperature at the cooler portion of the lake is ~48˚C.

Microbiology of BSL

The composition of the planktonic and sediment prokaryotic community of BSL

(at sites A and D) was explored by analyzing multiple 16S rRNA gene clone libraries

(116, 117). Due to the warmer and more variable conditions at site D versus site A, phylotype richness was expected to be lower at site D than at site A, and considerable differences were expected in terms of community composition. However, no significant reduction in phylotype richness was found in site D, and the communities at sites A and

D were surprisingly similar despite the 30˚C temperature differential. Convective mixing and gas inputs into the system seem to homogenize the communities in this system (117).

Archaeal phylotypes were numerically abundant in sediment samples while water samples were dominated by bacterial phylotypes. The four most prevalent phylotypes in the clone libraries derived from sediment samples shared an average of ~85% sequence identity to the closest cultivated Archaea representative, suggesting the presence of diverse novel organisms (116). The most prevalent phylotypes in the clone libraries from the water column (117) shared 99% sequence identity with Acidimicrobium sp. Y0018 isolated from Yellowstone National Park (57) and Hydrogenobaculum sp. Y04 ANCI.

Both phylotypes were also present as minor members in the sediment clone libraries. The closest cultured relative to Acidimicrobium sp. is the sole and type strain Acidimicrobium ferrooxidans ICPT (Genebank accession number CP001631), isolated from Icelandic hot

springs in the Krisuvik geothermal area (24). Members of the species have been isolated

or detected molecularly in warm, acidic, iron-sulfur-, or mineral-sulfide-rich environments (16, 24, 80). Acidimicrobium ferrooxidans is an autotrophic and

mixotrophic bacterium that is able to oxidize iron and grow anaerobically on Fe3+ with

the presence of an organic carbon donor.

Production in BSL

Recently, functional genes were assessed using functional gene clones libraries

and functional gene microarray from BSL site A water (102). Thirteen different primer

sets were used to target genes encoding for enzymes from 5 known carbon fixation

pathways; Calvin Cycle (RuBisCo forms I, II, and II), Reductive TCA (ATP citrate lyase

and citryl CoA lyase), Reductive Acetyl CoA (formyl tetrahydrofolate synthetase), and 3-

Hydroxypropionate/4-Hydroxybuturate (malonyl CoA lyase). Both cbbL primer sets

targeting RuBisCo I (k2f/v2r and 595F/1387R), amplified a sequence that shared 86-88%

nucleotide identity (94-96% amino acid identity) to Acidimicrobium ferrooxidans DSM

10331. An additional sequence was amplified with 595F/1378R only, sharing 88-89%

nucleotide identity with Acidithiobacillus caldus (97% amino acid).

Geochip analysis showed that the majority of the carbon cycling signal on the microarray was due to hybridization of BSL DNA to probes associated with heterotrophy

(102). BSL is limited by both inorganic and organic carbon, suggesting that microbial production is fueled by primary and secondary production. Heterotrophic and/or mixotrophic acidophiles are commonly found growing alongside chemoautotrophic primary producers in natural (57) and anthropogenic (42, 57, 84, 89) acidic environments.

Comparison with Acid Mine Drainage Sites

Studies of acid mine drainage (AMD) systems provide one of the best examples for understanding interactions among microbial communities in acidic habitats. Key players found in AMD systems can be categorized as iron oxidizers, sulfur oxidizers and heterotrophs (89). Autotrophic iron oxidizers are required to generate ferric iron to solubilize minerals (ie. pyrite) as well as provide organic carbon to stimulate growth of other players. Sulfur oxidizers grow optimally with added carbon sources, producing sulfuric acid to keep the pH at the optimum for the iron oxidizing acidophiles. The presence of heterotrophs aids in degrading small soluble metabolites produced by autotrophs that may be self-inhibitory (e.g. pyruvic and glycolic acid).

Similar ecological roles found in the microbial community of AMD systems may also occur in BSL. The presence of RuBisCo I from Acidimicrobium (102) along with the high prevalence of Acidimicrobium in BSL planktonic 16S rRNA clone libraries (11,

117), suggests that this organism may be an important primary producer in BSL. In this study, we isolated iron-oxidizing Acidimicrobium along with physiologically similar

Sulfobacillus sp. and heterotrophic Alicyclobacillus spp.. These three moderately thermophilic genera represent potentially different metabolic functions in the BSL ecosystem. By conducting physiological and metabolic characterization of these organisms, we will begin to understand their roles in this extreme environment. We determined optimal growth conditions for isolates and investigated the ability for some isolates to oxidize iron, pyrite, sulfur, and tetrathionate.

CHAPTER 2: MATERIALS AND METHODS

Media Preparation

Ferrous sulfate with trypticase soy broth (FeTSB) medium contained, per liter, 1.8

g (NH4)2SO4, 0.7 g MgSO4 x 7 H2O, 0.25 g TSB (BD Diagnostic Systems, USA), 10 g

FeSO4 x 7 H2O, distilled water, and was adjusted to pH 2.0 with 37 N H2SO4. Ferrous

sulfate and potassium tetrathionate with TSB broth (FeSo) contained mineral salts and

TSB described above, with the following modifications, per liter: reduction of FeSO4 x 7

H2O to 6.69 g, addition of 0.7 g K2S4O6 and pH adjusted to 2.5 with 37 N H2SO4.

Plates of FeTSB and FeSo were prepared by amending broth recipes described

above (55). Three separate solutions were prepared and adjusted to pH 2.5 with 37 N

H2SO4: TSB-mineral salts (in 740 ml), ferrous sulfate (6.69 g/L FeSO4 x 7 H2O in 50 ml

distilled water) and potassium tetrathionate (0.7 g/L K2S4O6 in 10 ml distilled water). The

autoclaved TSB-mineral solution was cooled to <40˚C, prior to the aseptic addition of

filter-sterilized (0.2 µm Micropore filters) ferrous sulfate and potassium tetrathionate

solutions. Agarose from a 10X solution that was also autoclaved separately and cooled to

~60˚C, was added to base medium of FeTSB or FeSo (post-autoclaving) to a final

concentration of 0.7% or 0.5% (v/v), respectively. Media were rapidly mixed and

distributed into sterile petri plates.

Overlay plates of FeTSBo and FeSo (55) were prepared similarly to the single

layer counterparts described above with the following amendments. FeTSBo and FeSo

are two-layered solid gels that contain acidophilic heterotroph Acidiphilium SJH in the

bottom layer. When the autoclaved TSB-mineral solution cools to <40˚C, Acidiphilium

SJH is added to a final concentration of ~106 cells/ml, mixed with the agarose solution

and aseptically distributed to thick petri plates at half the depth and allowed to solidify.

The top layer is then rapidly assembled and poured over the solid bottom layer. Prior to

inoculation into the bottom layer, Acidiphilium SJH (NCIMB 12826) was grown in liquid

medium containing, per liter, 6.96 g FeSO4 x 7 H2O, 0.7 g K2S4O6, 1.8 g galactose and

0.25 g TSB, at pH 2.5 and incubated at 30˚C for up to 3 days (56).

Liquid 1X PTYG medium contained, per liter, 0.25 g peptone, 0.25 g tryptone,

0.5 g yeast extract, 0.5 g dextrose, 0.6 g MgSO4 x 7H2O, 0.07 g CaCl2 x 2H2O, distilled water, and adjusted to pH 2.0 with 37 N H2SO4. Gelrite-solidified 1X PTYG medium was

prepared by adding 5 ml per liter each of 2 M MgCl2 and 0.5 M CaCl2 solutions to the

aforementioned liquid medium. A 2X gelrite solution was prepared separately and

adjusted to pH 2.9 with 37 N H2SO4. Post autoclaving, gelrite was added to PTYG base

medium (at 80˚C) to a final concentration of 0.8%. Media were rapidly mixed and

distributed into sterile petri plates.

Washed agarose/yeast extract (WAYE) medium contained, per liter, 0.2 g yeast

extract, 0.5 g MgSO4 x 7H2O, 0.15 g (NH4)2SO4, 0.1 g KCl, 0.01 g Ca(NO3)2, distilled

water, and adjusted to pH 2.5 with H2SO4 (55). A water-washed agarose solution (250

ml) was prepared and autoclaved separately; agarose (7.0 g) was soaked for approximately 30 minutes in 1 L distilled water with continuous mixing. The solution was allowed to settle for 15 minutes and the supernatant was decanted. The washed agarose was centrifuged 10,000 rpm (RC5C Sorvall Instruments DuPont fixed angle rotor

SA-600) and the agarose pellets re-suspended in 250 ml distilled water and sterilized by

autoclaving. After autoclaving, the yeast/salts and agarose solutions were combined. Five

ml of a 0.2 µm filter-sterilized ferrous sulfate solution (0.696 g FeSO4 x 7 H2O in 5 ml

distilled water at pH 2.0) was aseptically added to the combined medium prior to

distributing to petri plates.

Autotrophic, Heterotrophic, Mixotrophic, Pyrite, Sulfur, Sulfur/YE,

Sulfur/YE/trace Fe, Tetrathionate, Tetrathionate/YE, and YE media for Acidimicrobium

cultivation contained different amendments to the following basal mineral salts, per liter,

0.4 g MgSO4 x 7H2O, 0.2 g (NH4)2SO4, 0.1 g KCl, and 0.1 g K2HPO4. Autotrophic medium was composed of mineral salts amended with 13.9 g/l FeSO4 x 7H2O adjusted to

pH 1.7 with 37 N H2SO4 (24), Heterotrophic medium was composed of basal mineral salts amended with 0.01 g/l FeSO4 x 7H2O and adjusted to pH 2.0 with H2SO4 (24).

Mixotrophic medium was composed of mineral salts amended with 2.9 g/l FeSO4 x 7H2O and adjusted to pH 2.0 with 37 N H2SO4 (24). Yeast extract was aseptically added to

sterilized, cooled Heterotrophic and Mixotrophic media to a final concentration of

0.025% from a previously autoclaved 3% (wt/vol) solution in distilled water. Pyrite

medium contained mineral salts amended with 1% (wt/vol) ground rock pyrite and pH

adjusted to 2.0 with 37 N H2SO4. Sulfur medium contained mineral salts amended with

0.5% (wt/vol) elemental sulfur (S˚) and pH adjusted to 2.0 with 37 N H2SO4. Elemental

sulfur was sterilized by tyndallization (unpressurized heating at 100˚C for 1 hour each for

3 successive days). Sulfur/YE medium was prepared identically to Sulfur medium but

also contained 0.01%YE from previously autoclaved 3% (wt/vol) solution in distilled

water. Sulfur/YE/trace Fe medium was prepared identically to Sulfur/YE medium but

were amended with 0.036 mM FeSO4. Tetrathionate medium contained mineral salts

adjusted to 2.0 with 37 N H2SO4. After autoclaving, 5 mM K2S4O6 was added aseptically

to mineral salts with a 0.2 µm filter from a 100mM stock solution adjusted to 2.0 with 37

N H2SO4. The pH remained at 2.0. Tetrathionate/YE medium was prepared identically to

Tetrathionate medium, but also contained 0.01% YE from previously autoclaved 3%

(wt/vol) solution in distilled water. YE medium contained mineral salts adjusted to 2.0

with 37 N H2SO4. After autoclaving, 0.01% YE was added from previously autoclaved

3% (wt/vol) solution in distilled water.

All media were sterilized by autoclaving at 121˚C, 15 PSI for 20 minutes unless

noted otherwise.

Isolation of Sulfobacillus and Alicyclobacillus Strains

The work described in this section was done by former undergraduate Jennifer

Hampton and by my advisor Patricia Siering.

Sample collection

During the summer of 2009, sediment/water slurry samples were aseptically

collected from the shoreline at BSL site A and D (Fig. 2) as previously described (103,

116, 117). Duplicate ½ serial extinction dilutions (to 2.56 x 10-6) were prepared in sealed

Balch tubes on site into three different types of liquid media (FeTSB, FeSo, and

Acidimicrobium autotrophic liquid media) and incubated at 45˚C and 50˚C on return to

campus (approximately 8-10 hours post-collection); enrichment headspaces were

replenished with 0.2 µm filter-sterilized air every two weeks. Additionally, 100 µL of

undiluted and diluted (10-1-10-3) slurry samples from each site were plated onto FeTSB

and FeSo plates on site, and incubated in sealed plastic containers at 45˚C and 50˚C on

return to campus (approximately 8-10 hours post-collection).

Winogradsky column enrichments

Duplicate Winogradsky-type Ferrous Sulfate and Ferric Citrate enrichment columns were created in 200 mm x 25 mm glass test tubes from BSL sediment and water

(site A), and one of each type were incubated at room temperature in a sunny window, and at 45°C in the dark for 1.5-5 months prior to culturing attempts. Enrichment columns was prepared by amending BSL sediment with an equal volume of a 2X media solution prepared in BSL site water, and pH adjusted to 2.8 with 5 N H2SO4: (1) Ferrous Sulfate

(FeSO4) enrichment contained (final concentration in g/L)- 3.0 g (NH4)2SO4, 0.5 g

K2HPO4, 0.5 g MgSO4-7H2O, 0.1 g KCl, 0.01 g Ca(NO3)2, 44.22 g FeSO4-7H2O; and (2)

Ferric Citrate enrichment contained (final concentration in g/L)- 13.7 g ferric citrate, 5.6 g sodium lactate (60% solution), 2.5 g NaHCO3, 1.5 g NH4Cl, 0.6 g NaH2PO4, 0.1 g KCl,

10 ml each of Wolfes’s vitamin and mineral solutions (8).

FeTSB, FeTSBo, FeSo, FeSo and WAYE plates were inoculated in duplicate with

100 μl of material from each column listed above. The plates were incubated at 50°C

aerobically (in sealed plastic containers) and microaerobically using BD* Diagnostic

anaerobic jars (Mitsubishi AnaeroPak*-Microaero, Mitsubishi Gas Chemical, Company,

Inc.). Serial extinction dilutions of material from each type of enrichment column were

prepared by inoculating duplicate series of ½ serial extinction dilution tubes to a final

dilution of 2.56 x 10-6. For each enrichment type, three different liquid types of media were inoculated: FeSo, FeTSB and Autotrophic, and each series was incubated at 45°C and 50°C. Headspace was replenished weekly with 0.2 µm filter-sterilized air.

Selection and purification of isolates

Once significant growth was obtained from plates (Fig. 3), unique colony types

were selected for single colony isolation (SCI). These unique colony types were re- streaked for SCI until they appeared pure (single colony morphology per plate and single

cell morphology at 1000X phase contrast microscopy). When the most dilute growth-

positive tubes in a dilution series contained a single cell morphology type (as indicated by

1000X phase contrast microscopy), they were diluted again (1/2 dilutions to 2.5 x 10-6).

After three rounds of extinction dilution culturing, the culture was considered pure (Fig.

3). Streaking for single colony isolation was also attempted from extinction dilution

tubes. Information regarding maintenance and freezing of isolates can be found in the

appendix (App. A, B).

Slurry Sample

Winogradsky Column Spread -plated Enrichments

Serial Extinction Dilutions Spread- plated Serial Single Colony Isolation Extinction (SCI) Dilutions SCI

Check for Purity of Isolates

Figure 3. Flow chart schematic summarizing methods for isolation of Sulfobacillus and Alicyclobacillus strains. Individual slurry samples were collected from BSL sites A and D (Fig. 2). Samples from both sites were spread onto plates or diluted to extinction in liquid media (see methods). Duplicate Winogradsky column enrichments were made from amending Site A mud with enrichment media. Post-incubation, enrichment columns were inoculated onto a variety of solid media and diluted to extinction in liquid media. Cultures were purified by single colony isolation (SCI) or extinction dilution culturing, and purity was verified microscopically. The isolation of Sulfobacillus and Alicyclobacillus was accomplished by former undergraduate Jennifer Hampton under the direction of Dr. Patricia Siering in our laboratory.

Isolation of Acidimicrobium sp. from BSL

Sample collection

On June 25, 2010, water samples were aseptically collected from the shoreline at

BSL site A (Fig.2) as previously described (103, 116, 117). Temperature and pH were

recorded on site using Thermo-Orion290A Plus meter (Fisher Scientific, Pennsylvania,

PA). Sediment was allowed to settle, prior to cultivation efforts. Within two hours of

collection, site water was inoculated for enrichment or onto plates (Fig. 4). Samples for

enrichment were diluted (in sterile media) to 1/30, 1/100, 1/1000, and 1/5000 and

inoculated into Wheaton serum bottles containing either of the three liquid media

(Autotrophic, Heterotrophic, or Pyrite); final volumes were 15 or 30 ml, in 60 or 100 ml

bottles, respectively. FeSo and FeSo plates were inoculated by spread-plating with 100 µl

of undiluted, 1/10, 1/100, and 1/1000 dilutions (in sterile media) of sample. Bottles were

sealed, and all inoculated media were left at ambient temperature for approximately 24

hours, followed by incubation at 45˚C and 50˚C upon return to HSU. The headspace of all serum bottles was replaced weekly with air that was passed through a gas filter and

0.2 µm syringe filter. Plates were incubated in sealed plastic containers.

Isolation and purification

Morphology and relative cell densities (growth) were monitored weekly by phase

contrast microscopy (400X and 1000X phase contrast microscopy) over the course of two

months. Enrichments that showed growth in the most dilute bottle were diluted to

27 extinction by serial 1/10 dilutions (to 1 x 10-8 final dilution) into fresh media. All enrichments showing growth were spread-plated (100 µl) onto FeSo and FeSo, and incubated at source temperature of the enrichment.

Inoculated plates were observed daily for evidence of growth. Once significant growth was obtained, unique colony types were selected for SCI and were re-streaked until they appeared pure. Plates that indicated growth of a potential iron-oxidizing bacteria (red/orange encrusted colonies or smears) were screened via PCR with

Acidimicrobium-specific 16S rRNA primers AmcF/AmcR and universal primers (see II.

D.1). Information regarding maintenance and freezing of isolates can be found in the appendix (App. A, B).

Supernatant from Slurry Sample

Spread -plated Enrichments

Spread- plated Serial Single Colony Isolation Extinction (SCI) Dilutions SCI

Check for Purity of Isolates

Screen Orange Colonies/Smears by PCR (AmcF/AmcR)

Figure 4. Flow chart schematic of methods summarizing isolation of Acidimicrobium sp. Water from slurry sample collected from site A (Fig. 2) was inoculated onto plates and enrichment media (see Methods). Plates were also inoculated from all enrichments showing growth and incubated at enrichment source temperature. Serial extinction dilutions were started from the most dilute enrichment showing growth and incubated at source temperature. Cultures inoculated onto plates were streaked for purity by single colony isolation (SCI). Purity of isolates was verified by plate and cell morphology. Orange colonies/smears were screened via PCR with Acidimicrobium-specific SSU rRNA primers AmcF/AmcR (see Methods and Table 1).

Identification of Isolates by 16S rRNA Gene Sequencing

Acidimicrobium-specific 16S rRNA primer design

16S rRNA gene sequences derived from members of the Acidimicrobium genus

(in Actinobacteria phylum) were obtained from the Ribosomal Database Project

(http://rdp.cme.msu.edu/) and from the NCBI database (http://www.ncbi.nlm.nih.gov/).

Using the program Sequencher 4.8 (Gene Codes Corporation, Ann Arbor, MI), these sequences were aligned with sequences from the Acidimicrobium phylotype found in

BSL sites A and D (117). Additionally, sequences of other taxonomically-related species

(e.g. members of the Firmicutes phylum – Alicyclobacillus, Sulfobacillus, Geobacillus, and members of the Actinobacteria phylum – Arthrobacter, Microbacterium,

Staphylococcus) that have been detected and isolated from BSL (47, 116, 117) were imported and aligned in Sequencher with the Acidimicrobium sequences. This alignment was scanned for stretches of nucleotides which were fairly conserved in the

Acidimicrobium 16S genes, but different from the other taxonomically-related BSL dwellers. Fifteen possible primer sequences (some in the forward and some in the reverse direction) were obtained from this alignment. The Ribosomal Database project and

NCBI nucleotide BLAST were used to analyze the in silico specificity of each primer to the Acidimicrobium genus. Potential primers and primer pairs were analyzed in the program OligoAnalyzer 3.1

(http://www.idtdna.com/analyzer/applications/oligoanalyzer/) for their likelihood of forming primer dimers and hairpins, etc. We chose to use a previously published reverse

primer Amf:995 (25), subsequently called AmcR, with a forward primer (AmcF)

designed herein. AmcF and AmcR target positions 554-577, 995-974 (E. coli numbering), respectively (Table 1). The design and testing of these primers was conducted by former undergraduates Jennifer Hampton and Jonathon Schultz under the direction of Dr. Patricia Siering in our laboratory.

SSU rRNA gene amplification by PCR

Orange colonies and growth smears were screened by whole cell (single-colony)

PCR as putative Acidimicrobium isolates using primers (AmcF/AmcR). Isolates that were

positive for amplification with AmcF/AmcR primers were subjected to additional PCR

reactions to amplify the entire SSU rRNA gene using primer sets 8F/690R and

341F/U1406R (Table 1). Whole cell PCR using primer set U341F/U1406R was not

possible for Acidimicrobium-positive isolates. Instead, we collected cells grown in liquid

heterotrophic medium at 45˚C for ~1 week until the maximum cell density was reached.

One milliliter volumes of culture (2 ml total) were centrifuged at 10,000 x g for 5

minutes, decanted, and then resuspended with 1 ml nuclease-free water. Different

dilutions of resuspended cells were subjected to PCR amplification. Primers 8F/690R

and U515F/P1525R or U341F/U1406R (Table 1) were used to amplify the entire SSU rRNA genes for all putative Alicyclobacillus and Sulfobacillus isolates. For each putative

isolate, six individual colonies (or growth smears) from freshly cultivated plates were subjected to whole cell PCR amplification in separate 25 µL reactions. Reactions were prepared in MasterMix (Promega Corporation, Madison, WI), and amplified using

annealing temperatures ranging from 55-58˚C and one minute dwell times. Amplicon sizes and specificities were confirmed by agarose gel electrophoresis by comparison to known sized standards. A minimum of 3 positive reactions were pooled by volume prior to purification using Wizard® PCR preps DNA Purification Systems (Promega, Madison,

WI), per manufacturer’s recommendations.

Clone library construction and sequence analysis

Pooled and purified amplicons derived from single colonies of putative

Alicyclobacillus and Sulfobacillus isolates were sent to SagaGene of Palo Alto, California for direct sequencing of SSU rRNA genes. Pooled and purified amplicons of putative

Acidimicrobium isolates were cloned into the pGEM-T Easy cloning vector (Promega,

Madison, WI) and transformed into competent E. coli JM109 cells (Promega, Madison,

WI) per manufacturer’s recommendations. Plasmids from white clones were subjected to single colony PCR using vector primers T7F/M13R (Table 1) and confirmed for expected product size by agarose gel electrophoresis. Negative controls consisting of blue colonies were also screened as negative controls. Plasmid DNA was isolated from confirmed clones using Wizard® Plus SV Minipreps (Promega, Madison, WI), according to manufacturer’s recommendations. Concentrations of plasmid DNA were determined by diluting and comparison with known molecular weight standards on agarose gels, and spectrophotometrically (Thermo Scientific NanoDrop 1000 spectrophotometer,

Wilmington, DE). For each putative Acidimicrobium isolate, plasmids from fifteen clones from the AmcF/AmcR library and 13 clones from the U341/1406R library were

sequenced in both directions by Sagagene BioScience Corporation (Palo Alto, CA) with

the ABI3730xl genetic analyzer.

Sequences were analyzed and vector sequences were removed using the

Sequencher DNA sequence software 4.8 (Gene Codes Corporation, Ann Arbor, MI).

SDSC Biology Workbench 3.2 (http://workbench.sdsc.edu/) was utilized to perform

alignments of sequences to each other in Clustal W and to estimate genetic distances

using Clustal Dist (109). Sequence identity was compared to those in the NCBI database

(http://www.ncbi.nlm.nih.gov/) using the basic alignment local alignment search tool

(BLAST) for nucleotide sequences (4). We searched the Nucleotide collection (nr/nt) database using Megablast (optimized for highly similar sequences). Strain identification was accomplished with the help of former undergraduates Clayton Carey and Jonathon

Schultz under the direction of Dr. Patricia Siering.

Table 1. 16S rRNA primers utilized.

Primer Direction Sequence Reference U8F Forward 5’-AGAGTTTGATCCTGGCTCAG-3’ (63) U690R Reverse 5’-TCTACGCATTTCACC-3’ (63) U515F Forward 5′-GTGCCAGCAGCCGCGGTAA-3’ (92) P1525R Reverse 5’-AAGGAGGTGATCCAGCC-3’ (63) U341F Forward 5’-CCTACGGGRSGCAGCAG-3’ (44) U1406R Reverse 5’-GACGGGCGGTGTGTRCA-3’ (92) AmcF Forward 5’-GTCGGATGTGAAATCACCAGGCTC-3’ This work AmcR Reverse 5’-CTCTGCGGCTTTTCCCTCCATG-3’ (25) T7F Forward 5’-TACGACTCACTATAGGG-3’ Promega pGem T easy protocol M13R Reverse 5’-CAGGAAACAGCTATGAC-3’ Promega pGem T easy protocol

Morphology of Acidimicrobium by Electron Microscopy

Isolates EAO1, EAO2, EAO4 and the Acidimicrobium ferrooxidans ICPT (DSM

10331, type strain) were grown in Heterotrophic and Mixotrophic medium at 45˚C for 1

week until they reached late exponential/early stationary phase.

Transmission electron microscopy (TEM)

Exponential phase cultures were cooled to room temperature and 4 ml of culture

were spun down in a centrifuge at 12000 x g to pellet cells. Centrifuged tubes were

immediately placed in ice, supernatant was removed and pellet was resuspended in 1 ml

3% (V/V) glutaraldehyde in 50 mM cacodylate buffer-HCl (pH 7.0). Cells were fixed at

room temperature for 1h, then placed on ice for at least 15 min. Fixed cells were

harvested by centrifugation at 12000 x g, washed in 1 ml of 50 mM cacodylate buffer-

HCl (pH 7.0) buffer (3 times), and stained with 1% (vol/vol) osmium tetroxide in distilled

water. Cells were centrifuged and rinsed with 1 ml of distilled water (3 times), and

embedded in 4% agar blocks. The Pelco BioWave® 3451 lab microwave system (Ted

Pella Inc., Redding, CA) was utilized for dehydration and infiltration processes per manufacturer’s recommendations. Agar blocks were sliced and dehydrated in a graded series of increasing ethanol concentrations (25%, 50%, 75%, and 100%) at 40 seconds each (37˚C) using the microwave system. Resin ERL-4221 (3,4-Epoxycyclohexylmethyl

3,4-epoxycyclohexanecarboxylate) (Ted Pella, Inc., Redding , CA) was prepared using the standard block formulation, per manufacturer’s recommendation. Dehydrated sections were infiltrated in a series of increasing resin concentrations for 15 minutes each at 45˚C,

using the microwave system. The first resin mixture contained equal volumes of resin and

100% ethanol. Immediately following, sections were infiltrated into fresh resin, twice in

succession. Samples were transferred to BEEM capsules or molds and allowed to

polymerize overnight in a 60˚C oven. Ultrathin sections were cut on Reichert Om U2

ultramicrotome with a Diatome diamond knife and post-stained with uranyl acetate and

lead citrate (91). All samples were observed with a Phillips EM 208S transmission

electron microscope operated at 60 kV.

Scanning electron microscopy (SEM)

Four one-ml volumes of exponential phase cultures (grown approximately 1

week) were collected onto a 0.2 µm nucleopore membrane filter and immediately fixed in

3% (V/V) glutaraldehyde in 50 mM cacodylate buffer-HCl (pH 7.0). Cells were fixed at

room temperature for 1h. After fixation, cells were washed with 50 mM cacodylate

buffer-HCl (pH 7.0) and dehydrated in a graded series of increasing ethanol

concentrations (25%, 50%, 75%, and 3 times in 100%) for 10 minutes each. The center

of the membrane filter was cut and then processed through a critical point dryer (Polaron,

Watford, England) and sputter-coated with gold. All samples were observed with Topcon

ABT-32 scanning electron microscope from 25-30 kV and 6 mm working distance.

Physiological Characterization of Isolates

Preparation of inocula and growth assessment

Acidimicrobium sp., Sulfobacillus sp., and Alicyclobacillus sp. isolates obtained

from this work (Table 3) and A. ferrooxidans ICPT were examined for optimal growth

conditions and possible energy sources. For all growth experiments, inocula were

prepared by subculturing single colonies or smears (for Acidimicrobium isolates) from

fresh (1-5 days old) FeSo plates (for Acidimicrobium and Sulfobacillus), or 1X PTYG-

gelrite plates (for Alicyclobacillus sp.), into specified media, and incubating at 50˚C until

cultures were in exponential phase as determined by (microscopically) following

increases in cell number over time. Equal numbers of actively growing cells were

transferred, in duplicate, to experimental medium to an initial cell density of

approximately 5x103 cells/ml. All growth experiments, except those for determination of

temperature and pH range and optima, were conducted with media prepared at pH 2.0 and incubated at 50˚C. For experiments lasting longer than 5 days, the headspaces of tubes were replenished every 5 days with 0.2 µm filter-sterilized air. For growth experiments with Acidimicrobium and Sulfobacillus, relative growth was assessed by direct counts using a hemocytometer (400X phase contrast microscopy) at a minimum of three different time points during exponential growth. Differences in morphologies associated with variable growth condition(s), if any, were noted. Growth experiments with Alicyclobacillus were monitored by both direct cell counts and optical density at 595 nm using Bausch & Lomb Spectronic 20 (Rochester, NY) spectrophotometer.

The average of direct cell counts for each time point was used to first calculate n,

the number of generations during the period of exponential growth as:

Equation 1:

= 0 2 𝑛𝑛 𝑙𝑙𝑙𝑙𝑙𝑙𝑙𝑙 − 𝑙𝑙𝑙𝑙𝑙𝑙𝑁𝑁 𝑙𝑙𝑙𝑙𝑙𝑙 where N is the final cell number and N0 is the initial cell number. To determine doubling

time (or time/generation), the duration of exponential growth (t) expressed in hours was

divided by n.

Determination of pH and temperature optima and range for growth

Growth experiments. Inocula cultures and growth experiments for determination

of optimal temperature and pH were prepared in liquid Heterotrophic medium (pH 2.0)

for Acidimicrobium and Sulfobacillus, and in 1X PTYG liquid medium (pH 3.0) for

Alicyclobacillus. To determine temperature ranges and optima for growth of isolates,

duplicate tubes were incubated at 25˚C, 30˚C, 35˚C, 45˚C, 50˚C, 60˚C and 70˚C. To

determine pH ranges and optima, duplicates were inoculated into liquid medium adjusted

to pH 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0 (with H2SO4 and NaOH), and incubated at 50˚C

(for isolates obtained in this work) or 45˚C (A. ferrooxidans ICPT).

Linear regression models and Akaike information criterion (AICc). In order

to determine whether pH and temperature variables could distinguish among 11

Sulfobacillus isolates (Table 4) and/or could distinguish between species of

Acidimicrobium and Sulfobacillus, we performed a linear regression and ranked models

by AICc (73) with R software (version 2.15.1) (see Appendix equations for generating

AICc values). To compare various growth rates of isolates, we first calculated the number

of generations produced during exponential phase (see Eq.1). The division rate was used

as the response variable in the analysis, and this was calculated by dividing the number of

generations per unit of time in an exponentially growing culture. Regression and AIC

analyses and interpretation were conducted by Connor Fitzhugh, a graduate student in our

laboratory under the direction of Dr. Rob Van Kirk in the department of Mathematics at

HSU.

Iron and pyrite as energy sources

Autotrophic growth of Acidimicrobium sp. (EAO1 and EAO4 isolated in this

work, and A. ferrooxidans ICPT) were compared under varying iron concentrations with a

headspace of enhanced CO2 (3-5% v/v) or filter-sterilized air. Inocula cultures grown in

Autotrophic medium were sub-cultured into fresh Autotrophic medium containing the following iron concentrations; 50 mM, 25 mM, 10 mM, 2 mM, 0.68 mM, 0.136 mM, and

0 mM. Isolates obtained in this work were incubated at 50˚C, and the ICPT type strain

was incubated at 45˚C.

T Mixotrophic growth of EAO1, A. ferrooxidans ICP , JAO1, and JWO19m were

examined. Inocula cultures grown in Mixotrophic medium were sub-cultured into fresh

Mixotrophic medium, amended as follows: no YE or iron (mineral salts only), no iron

(0.01%YE only), no YE (10 mM FeSO4 only), heterotrophic (0.01% YE and 0.036 mM

FeSO4), and 0.01%YE amended separately with 1 mM, 10 mM, 25 mM or 50 mM

FeSO4. In addition, inocula cultures were sub-cultured into fresh Pyrite medium amended with 0.01%YE.

Iron and pyrite oxidation via ferrozine assay

Evidence of iron and pyrite oxidation was observed by comparing growth and

ferrous iron oxidation using the colorimetric ferrozine assay (66) which measures total

soluble ferrous iron. All assays were analyzed, in triplicate, using HP8452A diode array

spectrophotometer at 562 nm. Samples, controls and standard solutions were treated

identically.

Acidimicrobium isolates, A. ferrooxidans ICPT, and six Sulfobacillus isolates

(JAO1, JWO13, JWO19m, JWO20m, JWO21m, and JWO22m) were cultured in

Mixotrophic medium with YE amended to 0.01% and sub-cultured separately into

Mixotrophic and Pyrite media, with and without 0.01% YE. After growth, a 25 µL

sample of culture (cooled to room temperature) was combined with 5 mL of ferrozine (1

g/liter) in 50 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffer

at pH 7. Negative controls containing 2.5% (v/v) glutaraldehyde-killed EAO1 at

maximum cell density (106 cells/ml) and uninoculated media were also monitored for

2 abiotic iron oxidation. A calibration curve (R ≥0.9950) using Autotrophic media (at pH

2.0) as standard solutions and reagent/sample blank (basal salts only at pH 2.0) were

prepared and monitored for each time point. Ferrous iron concentration was measured

spectrophotometrically (562 nm) after complexation with ferrozine reagent within 2

hours of sampling/calibration preparation.

Pyrite dissolution (oxidation) was assessed by reducing the total soluble iron that

was present with 0.1 M hydroxylamine hydrochloride in 0.1 M HCl for 10 seconds prior

to combining sample with ferrozine/HEPES, as described above. Additional standards

were prepared as follows: calibration curve using FeCl3 in 0.5 M hydrochloric acid; 0.5

M HCl reagent blank; and negative control of unreduced 25 M FeCl3. Ferric iron

concentrations were calculated as the difference between total soluble iron and ferrous

iron.

Sulfur and tetrathionate oxidation

Sulfobacillus isolates (JAO1, JWO19m, JWO20m, JWO21m, JWO22m) ,

Acidimicrobium isolates (EAO1, 2 and 4), and A. ferrooxidans ICPT were assessed for

their ability to oxidize sulfur and tetrathionate. Inocula were prepared by culturing

Sulfobacillus isolates in Sulfur/YE media and Acidimicrobium in Heterotrophic medium.

Both genera were sub-cultured to either Sulfur media (with and without 0.01% YE),

Tetrathionate (with and without 0.01% YE), and YE media (0.01% YE). Additionally,

Acidimicrobium strains were inoculated into Heterotrophic medium, Heterotrophic medium adjusted to 0.01% YE (heterotrophic), and Sulfur/YE/trace Fe medium (0.036 mM FeSO4). Evidence of sulfur oxidation to sulfate was determined by measuring culture

pH. The pH of uninoculated controls was also monitored to control for abiotic

acidification.

CHAPTER 3: RESULTS

Isolation, Identification and Naming of Alicyclobacillus and Sulfobacillus Isolates

Twenty isolates were obtained from plated samples of Winogradsky enrichments

or from BSL site A spread plates (Table 2). No isolates were obtained from serial

extinction dilutions. Eleven isolates were obtained from the FeSO4 Winogradsky enrichments, eight of which (JWO10, JWO11, JW012, JWO13, JWO16m, JWO17m,

JWO18m, and JWO19m) were obtained from columns incubated at room temperature by a

sunny window, and 3 isolates (JWO20m, JWO21m, JWO22m) were obtained from columns incubated at 45˚C in the dark. Regardless of enrichment incubation temperature, all isolates obtained from FeSO4 Winogradsky enrichments were obtained by sub-

culturing enrichment onto FeSo at 50˚C for 5-10 days. Seven of the isolates were

obtained from microaerobic incubations (JWO16m - JWO22m) while the others were

obtained from aerobic incubations (JWO10, JWO11, JWO12, and JWO13). All isolates

from the FeSO4 Winogradsky enrichments produced round ‘fried egg’ colony

morphologies. The ‘fried egg’ colonies appeared as dark orange growth in the center of

the colony surrounded by white growth (Fig. 5a). The cell morphology of these isolates

ranged from thick (0.5-0.8 µm thick) single/paired rods to filamentous (not shown). All

the above-described Winogradsky isolates produced spherical terminal endospores (not

shown).

The two isolates (JWW6 and JWO15) obtained from ferric citrate Winogradsky

enrichments were from columns that were incubated aerobically at room temperature by a

sunny window. Isolate JWW6 was obtained by sub-culturing onto WAYE at 50˚C for 3-5 days. JWW6 produced round white colonies consisting of single and paired rods (~0.5µm thick) with oval terminal endospores. Isolate JWO15 was sub-cultured onto FeSo at 50˚C

for 5-10 days. JWO15 produced round ‘fried egg’ colonies comprised of motile single

and filamentous rods (not shown) with spherical terminal endospores.

Seven isolates (JAO1, JAO2, JAO3, JAO4, JAW5, JAW7, and JAW8) were

obtained from direct plating of BSL samples onto FeSo solid media with aerobic

incubation at 50˚C for 3-5 days. Isolates JAO1-4 produced either ‘fried egg’ or round

orange colony morphologies containing cells as single, paired, or filamentous rods with

spherical terminal endospores. Single rods were about 0.5-0.8µm in diameter, and the

length was 1.6µm and longer (Fig. 6a). Isolates JAW5, JAW7, and JAW8 produced

white/off-white colonies. JAW5 and JAW7 produce similar white, defined margin

colonies consisting of single, paired or short chains of rod-shaped cells (~0.8µm thick)

(not shown). JAW8 produces irregular flat off/white colonies, and cells consistently

appear as slightly longer chains than JAW5 and JAW7 (Fig 6b). All three JAW isolates

produce oval endospores at the terminal end (not shown).

SSU rRNA genes from isolates JAO1 - JAW8 were amplified with

U341F/U1406R, and those from JWO10 - 15 and JWO16m - JWO22m were amplified using 8F/690R and 515F/1525R and used to generate a single contig that corresponded to the near-complete 16S rRNA gene. Sequence analyses identified 16 isolates as

Sulfobacillus acidophilus (BLAST ID 99%) and 4 as Alicyclobacillus tolerans or

Alicyclobacillus sp. DSM 6481 (99%) (Table 2). The sixteen isolates closely related to S.

43 acidophilus all produced orange ‘fried egg’ colony morphologies. All isolates closely related to Alicyclobacillus sp. produced white colonies. All twenty isolates were Gram stained and appeared Gram variable or Gram positive (1000X oil immersion microscopy)

(Fig. 6a, b).

Each isolate obtained was identified by a four digit code that describes isolation source and morphology XXX#. The first letter signifies isolation attempts by Jennifer

Hampton (J). The second letter indicates the source of isolation such as BSL site A (A) or

Winogradsky enrichment (W). The third letter describes the color of the colony morphology as orange (O) or white (W). The number at the end is the numerical order that the isolates were obtained for the attempted isolation series. The ‘m’ subscript identifies organisms that were isolated under microaerobic conditions.

Isolation, Identification and Naming of Acidimicrobium Isolates

At the time of sampling, BSL site A water was 50.8˚C and pH 2.37. Three EAO strains were isolated at 45˚C from a combination of serial extinction dilutions and streak plates derived from enrichments in 1% pyrite or Autotrophic medium (Table 2). After

~14 days, both enrichments were turbid and yellow, indicating iron oxidation. EAO1 and

EAO4 were obtained from dilutions of 1% pyrite enrichment streaked onto FeSo and

FeSo, respectively. Enrichment cultures contained numerous thin (diameter <0.5 µm) rods of varying length that lacked endospores. When enrichments were sub-cultured on ferrous sulfate plates for 6-14 days, no single colonies were observed. Instead, dark

orange smears that contained cells of similar morphology to those observed in previous

enrichments were found (Fig. 5b).

EAO2 was obtained from Autotrophic liquid medium enrichment (1/5000) sub- cultured onto FeSo plates. The enrichment contained mobile thin (< 0.5 µm diameter),

short and filamentous rods devoid of endospores (Fig. 6c). Plate growth after 6-14 days

incubation also appeared as bright orange smears instead of single colonies, and the smears contained similar cell morphologies as were observed in the enrichment. Orange smears from EAO1, EAO2 and EAO4 were positive for Acidimicrobium when screened

by PCR with AmcF/AmcR primers (Table 1).

No growth was observed after two rounds of serial extinction dilutions from either

medium type. Although there was growth in Heterotrophic 50˚C enrichments (containing

various sized rods), I was unable to obtain growth on plates after subculturing. We also

inoculated activated carbon plates that were prepared the same as FeSo and FeSo, but

contained 2% charcoal in both layers. We postulated that the addition of charcoal may

enhance selection of iron-oxidizing autotrophs by adsorbing inhibitory metabolites (e.g.

organic acids) produced during autotrophic growth. Activated carbon plates produced

comet shaped growth smears but could not be further sub-cultured. In addition to EAO1,

EAO2, and EAO4, thirteen other “isolates” were followed. These other thirteen isolates varied in colony morphologies from round translucent/white to yellow streaks/comets.

Since these isolates were negative for amplification with Acidimicrobium specific 16S rRNA primers AmcF/AmcR, we discontinued working with them.

SSU rRNA genes of EAO isolates were amplified using both primer sets

U341F/U1406R (~1100nt) and AmcF/AmcR (450nt) (Table 1). The 16S rRNA short sequence amplified by the AmcF/AmcR primer set aligns in the middle of the longer sequence amplified by the universal primer set over the 313-737 bp region. Analysis of sequences from both primer sets for EAO1, EAO2, and EAO4 showed 99% 16S rRNA gene sequence identity to Acidimicrobium sp. Y0018, A. ferrooxidans ICPT, and to each other over the entire length analyzed (Table 2). Acidimicrobium sp. Y0018 is a phylotype previously identified in SSU rRNA libraries prepared from BSL derived DNA (117).

Each isolate was identified by a four digit code that describes isolation source and morphology XXX#. The first letter represents the isolation method of either enrichment

(E) or spread plating (P). The second letter indicates the source of isolation such as BSL site A (A). The third letter describes the color of the colony morphology as orange (O) or white (W). The number at the end is the numerical order that the isolates were obtained for the attempted isolation series.

Table 2. 16S rRNA gene sequence analysis, isolation conditions, and morphology of isolates. Identity of isolates was determined by BLAST analysis of near-complete 16S rRNA genes over 1100 nta or 1500 ntb (4). Colony morphologies were observed during single colony isolation onto FeSo (Acidimicrobium/Sulfobacillus) or 1X PTYG (Alicyclobacillus). All isolates are rods and size was measured from images taken by electron microscopy (EM) or phase contrast (PC). Production of endospores was indicated as either absent (-) or present (+) as spherical endospores (s), oval endospores (o), and position of spore on the terminal end (t). Gram stain results were either positive (+), negative (-) or variable (v). Additional abbreviations include; room temperature (RT), extinction dilutions (ED), and not determined (ND). %Sequence Enrichment/Isolation Cell morphology Produce Gram Isolate Most related organisms Colony morphology similarity conditions (dilution) (diameter µm) endospores stain EAO1 Acidimicrobium sp. Y0018, 99a 1% pyrite enrichment Orange smears Single, paired, - + A. ferrooxidans ICPT 45˚C/ 1st series ED (10-1), filamentous (0.3-0.4 FeSo 45˚C EM) EAO2 Acidimicrobium sp. Y0018, 99a Autotrophic enrichment Orange smears Single, paired, - + A. ferrooxidans ICPT 45˚C/ FeSo 45˚C filamentous (0.3-0.4 EM) EAO4 Acidimicrobium sp. Y0018, 99a 1% pyrite enrichment Orange smears Single, paired, - + A. ferrooxidans ICPT 45˚C/ 1st series ED (10-1), filamentous (0.3-0.4 FeSo 45˚C EM) JAO1 Sulfobacillus acidophilus 99a FeSo 50˚C Round fried egg Single, paired (0.8 +(s/t) +/v PC) JAO2 Sulfobacillus acidophilus 99a FeSo 50˚C Round orange Single, paired (0.5 +(s/t) ND EM) JAO3 Sulfobacillus acidophilus 99a FeSo 50˚C Round fried egg Paired +(s/t) ND

JAO4 Sulfobacillus acidophilus 99a FeSo 50˚C Round fried egg Filamentous +(s/t) +/v

JAW5 Alicyclobacillus tolerans 99a FeSo 50˚C Round white Single, paired, short +(o/t) v chained (0.8 PC) JWW6 Alicyclobacillus sp. DSM 99a Winogradsky Ferric Round white Singe, paired (0.8 +(o/t) v 6481 Citrate RT/ WAYE 50˚C PC)

JAW7 Alicyclobacillus tolerans 99a FeSo 50˚C Round off-white Single, paired (0.8 +(o/t) v PC) JAW8 Alicyclobacillus tolerans 99a FeSo 50˚C Irregular flat white Chains (0.8 PC) +(o/t) v

b JWO10 Sulfobacillus acidophilus 99 Winogradsky FeSO4 RT/ Round fried egg Filamentous +(s/t) ND FeSo 50˚C b JWO11 Sulfobacillus acidophilus 99 Winogradsky FeSO4 RT/ Round fried egg Filamentous +(s/t) ND FeSo 50˚C b JWO12 Sulfobacillus acidophilus 99 Winogradsky FeSO4 RT/ Round fried egg Filamentous +(s/t) ND FeSo 50˚C b JWO13 Sulfobacillus acidophilus 99 Winogradsky FeSO4 RT/ Round fried egg Single, paired, +(s/t) +/v FeSo 50˚C filamentous (0.5-0.8 PC) JWO15 Sulfobacillus acidophilus 99b Winogradsky Ferric Round fried egg Filamentous +(s/t) ND Citrate RT/ FeSo 50˚C b JWO16m Sulfobacillus acidophilus 99 Winogradsky FeSO4 RT/ Round fried egg Single, paired (0.5- +(s/t) +/v FeSo 50˚C 0.8 PC) b JWO17m Sulfobacillus acidophilus 99 Winogradsky FeSO4 RT/ Round fried egg Paired, filamentous +(s/t) +/v FeSo 50˚C (0.5-0.8 PC) b JWO18m Sulfobacillus acidophilus 99 Winogradsky FeSO4 RT/ Round fried egg Single, paired (0.5- +(s/t) +/v FeSo 50˚C 0.8 PC) b JWO19m Sulfobacillus acidophilus 99 Winogradsky FeSO4 RT/ Round fried egg Single, paired (0.5- +(s/t) +/v FeSo 50˚C 0.8 PC) b JWO20m Sulfobacillus acidophilus 99 Winogradsky FeSO4 Round fried egg Single, paired (0.5- +(s/t) +/v 45˚C/ FeSo 50˚C 0.8 PC) b JWO21m Sulfobacillus acidophilus 99 Winogradsky FeSO4 Round fried egg Single, paired (0.5- +(s/t) +/v 45˚C/ FeSo 50˚C 0.8 PC) b JWO22m Sulfobacillus acidophilus 99 Winogradsky FeSO4 Round fried egg Single, paired (0.5- +(s/t) +/v 45˚C/ FeSo 50˚C 0.8 PC) (Table 2 continued)

a) b)

Figure 5. Comparison of colony morphology between Sulfobacillus and Acidimicrobium isolates. Sulfobacillus JWO18m (a) as well as all other Sulfobacillus isolates readily produces single colonies with a ‘fried egg’ morphology when cultured on FeSo plates. On the contrary, Acidimicrobium EAO isolates, represented by EAO2 (b), have difficulty forming single colonies and instead produce flat orange “smears” when streaked onto FeSo plates. The orange pigment is indicative of iron-oxidizing bacteria in which the ferrous iron present in the agar is oxidized to ferric iron and precipitated onto the agar’s surface.

a) Sulfobacillus JAO1 b) Alicyclobacillus JAW8

c) Acidimicrobium EAO2 d) A. ferrooxidans ICPT

Figure 6. Gram stained photographs of representative Sulfobacillus isolates (a), Alicyclobacillus isolates (b), and Acidimicrobium isolates (c) and A. ferrooxidans ICPT (d) viewed by 1000X light microscopy. Sulfobacillus and Acidimicrobium isolates/type strain were grown on FeSo plates at 50˚C for 2-4 days. Alicyclobacillus isolates were grown on 1X PTYG pH 2.9 at 50˚C for 2 days. All Sulfobacillus isolates (not shown) had similar cell morphologies to JAO1. Alicyclobacillus isolates (not shown) are similar to JAW8. Acidimicrobium EAO isolates have similar morphologies to EAO2. Small gray circles found on all images are dust particles on microscope (i.e. when mechanical stage is moved, spots remain). Size bar = 5µm.

Electron Microscopy of Acidimicrobium

Acidimicrobium EAO isolates had similar cell morphologies/ultrastructures to one another and to A. ferrooxidans ICPT (Fig. 7, 8), however the type strain produced slightly thinner (0.3-0.35 µm) rods than EAO isolates (0.3-0.4 µm) and occurred more frequently as filaments (not shown). Acidimicrobium cells occur most often as single or paired straight thin rods and do not form endospores (Fig. 7). Single rods are 1-2 µm long. There was no apparent difference in cell width between cells grown in Heterotrophic as compared to Mixotrophic medium (Fig. 7, 8). Filamentous cell morphologies were also observed, but less frequently under heterotrophic conditions. Cells stain Gram positive

(Fig. 6c, d), but areas that contain large amounts of ferric iron precipitate appear clear to negative (not shown). Actively growing cells in all media types appear motile under phase contrast microscopy even though they appear to lack flagella or pili in EM images.

They appear to move using a serpentine swimming motion (undulation) to propel them through the water.

The cell envelope appears to be composed of a layered cell wall ~ 30-60 nm thick

(Fig. 7) within a periplasm-like region, rather than a solid thick wall characteristic of

Gram positive bacteria (68). The cell membrane is clearly identifiable and is 7-8 µm thick. Although the outer surface appears electron dense, no outer membrane was evident at any magnification. Evidence for 0.1 µm long protrusions from the cell envelope were observed in some of the TEM images (Figs. 7b and d), and in some cases (Fig. 7d), these protrusions appear to connect cells. Other visible ultrastructures included small inclusions

(~80 nm diameter), ribosomes, nucleoid region and a single large inclusion (~150 nm diameter) or poly-β-hydroxybutyrate (PHB) granule. Mesosome artifacts were not

observed. Although the fixative was at pH 7.0, no evidence of lysis during fixation was

evident in EM images.

In scanning and transmission electron micrographs of Acidimicrobium EAO1, 2, 4

and A. ferrooxidans ICPT, large quantities of unknown particulates were observed (SEM

images Fig. 7a, 8a, TEM images not shown). These particulates range from 50-100 nm

in diameter and occur singly or aggregated. Characterized by thick dark surfaces, these

particulates resemble nanoparticles such as iron oxides or carbon filaments (77).

However, the hollow or lighter interior is not characteristic of oxides. These particulates

also appear as phase dense under phase contrast microscopy in both Heterotrophic and

Mixotrophic media (not shown).

a) b)

1.0µm 0.1µm

c) d)

R P N

0.5µm

I W M 0.5µm

Figure 7. Electron micrographs of Acidimicrobium EAO4 in Heterotrophic medium. Individual cells are 0.3-0.4 µm thick and 1-2 µm long and occur as (a) single, paired, or filamentous rods. The cell wall (W) is 30-60 nm thick with layering (c) and protrusions indicated by arrow (b and d). Visible ultrastructures are shown in (c); bilayer membrane (M), ribosomes (R), inclusions (I), nucleoid region (N), and potential single large poly- β-hydroxybutyrate (P) (or other type of ) inclusion granule. Similar structures were seen in other EAO isolates and ICPT strain. White arrow in image (a) shows unidentified nanoparticles present in media. Scale of each image is represented by length of size bars in microns.

b) a) b)

c) d)

Figure 8. Cell morphology comparison of Acidimicrobium EAO2 (left) and A. ferrooxidans ICPT (right) in Mixotrophic medium. EAO2 (a, c) has a slightly thicker diameter of 0.4 µm than the type strain at 0.35 µm. Cells in Mixotrophic and Heterotrophic medium have similar thickness. However, filamentous cells are more frequent in Mixotrophic medium. Results for EAO2 are similar for all EAO isolates (not shown). White arrow in image (a) shows unidentified nanoparticles present in media. SEM micrographs (a, b) contain 1.0 µm size bars and TEM micrographs (c, d) have 0.5 µm size bars.

Physiological Characterization of Isolates

Determination of temperature and pH range and optima

Growth experiments. Most Sulfobacillus isolates grew at 45-50˚C (Fig. 9a),

while three (JAO1, JWO16m, and JWO20m) were able to grow up to 60˚C (Fig. 9b), and

JWO22m was able to grow as low as 35˚C (Fig. 9c). The minimum pH for most

Sulfobacillus isolates was 2.0 (Fig. 10a), however JWO21m was capable of growth at pH

1.0 (Fig. 10b). Half of the isolates (JWO16m-21m) showed a distinct optimal pH of 2.0.

The other half (JAO1, JAO2, JWO12, JWO13, JWO18m, and JWO22m) had an optimal

pH between 2.0 and 3.0 (i.e. pH 2.5) (Fig. 10c). Collectively, Sulfobacillus isolates

obtained in this study were observed to grow in Heterotrophic medium from 35-60˚C and

pH 1-5, with an optimum of ~50˚C and pH 2.0-2.5. The optimal doubling time for all

Sulfobacillus isolates ranged from 3-7 hrs and maximum cell densities in Heterotrophic

medium were 5.20x105-8.08x106 cells/ml. Growth results for Sulfobacillus isolates are

summarized in Table 3.

Each Alicyclobacillus isolate had a unique physiology with respect to temperature

and pH preferences. Alicyclobacillus tolerans isolates JAW8 and JAW7 had a similar

permissible temperature range for growth (30-50°C) (Fig. 11a), but had different pH

ranges and optima. JAW8 had a narrower pH range (3.0-4.0) and lower optimum (pH

3.0) than JAW7 (range 2.0-4.0; optimum pH 4.0) (Fig. 12a, b). Alicyclobacillus sp.

JWW6 had the highest permissible temperature range for growth of 45-60°C (Fig. 11b).

JWW6 was also the only Alicyclobacillus isolate to have a permissible growth pH of 5.0

(range 3-5) (Fig. 12c). There was no notable difference in growth rates and yields at pH

3.0 and pH 4.0. The maximum cell densities at 50°C and either pH 3.0 or 4.0 were

7.71x107 cells/ml or 8.44x107 cells/ml, respectively. The doubling time of 0.8 hr was the

same at both pH values. The temperature and pH range for JAW5 were more restrictive

than was observed for other strains, with growth only occurring at 45-50°C (Fig. 11c), pH 3.0 (Fig. 12d).

As a group, the permissible growth temperature and pH ranges of Alicyclobacillus in 1X PTYG medium were 30-60˚C and 2.0-5.0, respectively. Alicyclobacillus isolates had a higher pH optimum (pH 3.0-4.0) than the other two genera, but maintained a similar optimum temperature (~50˚C). Alicyclobacillus isolates reached the highest cell densities (range of 2.46 x107-8.44x107 cells/ml) and maintained the fastest doubling time

(range of 0.8-2.2 hrs) compared to the two other genera examined in this study. Growth

results for Alicyclobacillus isolates are summarized in Table 3.

All Acidimicrobium isolates obtained in this study isolates displayed a narrow

permissible temperature range for growth of 45-50˚C (Fig. 13a). EAO2 and EAO4 were

capable of growth in pH 2.0-4.0 (Fig. 14a), while EAO1 was capable of growth from pH

2.0-5.0 (not shown). The optimal temperature and pH for all EAO isolates were 50˚C and

pH 2.0. Optimal doubling times in Heterotrophic medium for EAO isolates ranged from

6.3-7.3 hrs and maximum cell densities ranged 3.24x106-3.32x106 cells/ml (Table 3).

Compared to the EAO isolates, A. ferrooxidans ICPT type strain had a wider

permissible temperature range for growth (30-50˚C) and similar pH range 2.0-4.0 (Fig.

13 and 14). Optimal temperature and pH were 45-50˚C and pH 2.0. The type strain had an average doubling time ~6 hrs and reached 10X higher cell densities (avg. 4x107

cells/ml) than all the EAO isolates obtained in this study. However, maximum cell

densities at pH 2.0 and 3.0 may have been even higher since growth curves were not

followed into stationary phase (Fig. 14b). Growth results are summarized in Table 3.

1.00E+06 a) JAO2 b) JWO20m 20°C 1.00E+07 30°C 35°C 1.00E+06 45°C 1.00E+05 50°C

60°C 1.00E+05 cells / ml cells

70°C cells / ml

1.00E+04

1.00E+03 1.00E+03 0 10 20 30 40 50 60 70 80 0 20 40 60 80 100 120 timetime/hrs (hr) time (hr) c) JWO22m 1.00E+06 Figure 9. Three patterns of temperature growth for Sulfobacillus isolates represented by JAO2 (a), JWO20m (b), and JWO22m (c). Duplicates of each isolate were grown in Heterotrophic medium at pH 2.0, across a range

1.00E+05 of incubation temperatures. The average cell density at each time point was plotted. All Sulfobacillus isolates were

able to grow at 45-50˚C and optimally at 50˚C.

cells / ml cells Temperature growth curves of JWO12, JWO13, JWO17m,

1.00E+04 JWO18m, JWO19m, and JWO21m were similar to JAO2. Three isolates (JAO1, JWO16m and JWO20m), represented by JWO20m, were able to grow at 60˚C. JWO22m was the only Sulfobacillus isolate capable of growing at 35˚C.

1.00E+03 Lines are only shown for conditions that were permissive 0 10 20 30 40 50 60 70 80 for growth. Error bars represent standard error. timetime/hrs (hr) 57

a) JWO16m b) JWO21m pH 0.5 1.00E+06 1.00E+07

pH 1.0 pH 2.0

pH 3.0 1.00E+06 pH 4.0 pH 5.0 1.00E+05 pH 6.0

1.00E+05

cells / ml cells cells / ml cells

1.00E+04

1.00E+03 1.00E+03 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 timetime/hr (hr) timetime / (hr)hr c) JWO18m Figure 10. Three patterns of pH ranges and optima for 1.00E+07 Sulfobacillus isolates. Duplicates of each isolate were incubated at 50˚C and grown in Heterotrophic medium at different pH. The average cell density at each time point was plotted. Collectively, 1.00E+06 Sulfobacillus isolates grow at a range of pH 1.0-5.0 with an optimum of 2.0-2.5. The minimum pH for most isolates is 2.0 as 1.00E+05 represented by JWO16m (a). Only JWO21m (b) was able to grow / ml cells at pH 1.0. The optimal pH for half the isolates is closer to 2.0 (as shown by JWO16m and JWO21m) and the other half is between pH 1.00E+04 2.0-3.0, represented by JWO18m (c). Lines are only shown for conditions that were permissive for growth. Error bars represent standard error. 1.00E+03 0 10 20 30 40 50 60 70 80 timetime /(hr) hr 58

a) JAW8 b) JWW6 1.00E+08 1.00E+08 20˚C 30°C 35°C 45°C 1.00E+07 50°C 1.00E+07 60°C 70°C

1.00E+06

1.00E+06 cells / ml cells cells / ml 1.00E+05

1.00E+05 1.00E+04

1.00E+04 1.00E+03 0 20 40 60 80 100 0 5 10 15 20 25 30 35 40 45 timetime/hr (hr) timetime/hr (hr) c) JAW5 Figure 11. Three patterns for permissible temperature range of 1.00E+08 growth for Alicyclobacillus isolates. Duplicates were grown in

1X PTYG medium at pH 3.0 with varying incubation 1.00E+07 temperatures. All Alicyclobacillus isolates had an optimum temperature for growth of 50°C, but had different permissible temperature ranges of growth. Isolate JAW8 (a) and JAW7 (not 1.00E+06 shown) had the widest temperature range of 30-50°C. Isolate cells / ml cells JWW6 had the hottest temperature range 45-60°C (b). The 1.00E+05 narrowest temperature range (45-50°C) was observed for JAW5 (c). Results from direct cell counts and optical density readings (not shown) yielded similar results, but optical density 1.00E+04 measurements required incubation periods twice as long as direct counts. Lines are only shown for conditions that were permissive 1.00E+03 for growth. Error bars represent standard error. 0 5 10 15 20 25 timetime/hr (hr) 59

1.00E+08 a) JAW8 1.00E+08 b) JAW7 pH 1.0 pH 2.0

pH 3.0 1.00E+07 1.00E+07 pH 4.0

pH 5.0

pH 6.0 1.00E+06 1.00E+06 cells / ml cells pH 7.0 / ml cells

1.00E+05 1.00E+05

1.00E+04 1.00E+04 0 20 40 60 80 100 0 10 20 30 40 50 60 70 80 timetime /(hr) hr timetime/hr (hr)

1.00E+09 c) JWW6 1.00E+08 d) JAW5

1.00E+08 1.00E+07

1.00E+07

1.00E+06

cells / ml cells / ml cells 1.00E+05

1.00E+05

1.00E+04 1.00E+04

1.00E+03 1.00E+03 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 25 timetime/hr (hr) timetime/hr (hr) Figure 12. Unique patterns for pH range and optima for Alicyclobacillus isolates. Duplicates were incubated at 50˚C in 1X PTYG medium at varying pH. Isolate JAW8 (a) was able to grow from pH 3.0-4.0 with an optimum closer to pH 3. Only JAW7 (b) was able to grow at pH 2.0 (range 2.0-4.0) with an optimum ~pH 4.0. JWW6 (c) had the highest pH range (3.0-5.0) with an optimum pH 3.0-4.0. Isolate JAW5 (d) was observed to grow at pH 3.0 only. Results from direct cell counts and optical density readings (not shown) yielded similar results. Lines are only shown for conditions that were permissive for growth. Error bars represent standard error. 60

a) Acidimicrobium EAO2 b) A. ferrooxidans ICPT 20°C 1.00E+07 1.00E+08 30°C 35°C 45°C 1.00E+07 50°C 1.00E+06 60°C 70°C 1.00E+06

1.00E+05 cells / ml cells cells / ml cells 1.00E+05

1.00E+04 1.00E+04

1.00E+03 1.00E+03 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 time/hrtime (hr) timetime/hr (hr) Figure 13. Comparison of temperature range and optima between Acidimicrobium isolate EAO2 and A. ferrooxidans ICPT. For temperature ranges/optima, duplicates of each isolate/strain were grown in Heterotrophic medium at pH 2.0 with varying incubation temperatures. As represented by EAO2 (a), all Acidimicrobium isolates were observed to grow within a narrow temperature range (45-50°C), but had a clear optimum at 50°C. The type strain (b) had a wide permissible temperature range for growth (30-50°C) with an optimum between 45-50°C. Lines are only shown for conditions that were permissive for growth. Error bars represent standard error.

a) Acidimicrobium EAO2 b) A. ferrooxidans ICPT 1.00E+07 1.00E+08 pH 1.0 pH 2.0 pH 3.0 1.00E+07 pH 4.0 1.00E+06 pH 5.0 pH 6.0 1.00E+06

1.00E+05 cells / ml cells cells / ml cells 1.00E+05

1.00E+04 1.00E+04

1.00E+03 1.00E+03 0 20 40 60 80 100 0 20 40 60 80 100 120 140 160 180 time/hrtime (hr) time/hrtime (hr)

Figure 14. Comparison of pH range and optima between Acidimicrobium isolate EAO2 and A. ferrooxidans ICPT. For pH range/optima, duplicates were incubated at 45˚C (type strain) or 50°C (EAO isolates) with varying media pH. The pH range for EAO2 (a), EAO4 (not shown), and ICPT type strain (b) was 2.0-4.0 with an optimum at pH 2.0. Isolate EAO1 (not shown) had a slightly wider pH range of 2.0-5.0. Lines are only shown for conditions that were permissive for growth. Error bars represent standard error.

Table 3. Summary of temperature and pH ranges and optima for Acidimicrobium, Sulfobacillus, and Alicyclobacillus isolates. Each organism was inoculated, in duplicate, into media at different pH while incubated at a constant temperature and into media at a constant pH while incubated across a range of temperatures (see Methods). Ranges indicate minimum and maximum temperatures/pH that growth was observed (average of duplicates). Doubling time is the measure of time required for the cell population to double during exponential phase (see Methods). Temperature and pH conditions that contain both the fastest doubling time and highest maximum yield (cells/ml) are the optimal (opt.) growth conditions. Opt. Maximum Temperature Organism pH range (opt.) doubling cells/ml range ˚C (opt.) time (~hr) (at pH/°C) A. ferrooxidans ICPT 30-50 (45-50) 2.0-4.0 (~2.0) 6.5 2.06 x 107 (2/50) Acidimicrobium sp. EAO1 45-50 (~50) 2.0-5.0 (~2.0) 6.3 3.24 x 106 (2/50) Acidimicrobium sp. EAO2 45-50 (~50) 2.0-4.0 (~2.0) 6.4 3.32 x 106 (2/50) Acidimicrobium sp. EAO4 45-50 (~50) 2.0-4.0 (~2.0) 7.3 3.31 x 106 (2/50)

S. acidophilus JAO1 45-60 (50-60) 2.0-5.0 (2.0-3.0) 3.8 1.37 x 106 (2/60)

S. acidophilus JAO2 45-50 (~50) 2.0-5.0 (2.0-3.0) 3.8 1.07 x 106 (3/50)

S. acidophilus JWO12 45-50 (~50) 2.0-5.0 (2.0-3.0) 3.8 7.10 x 105 (3/50)

S. acidophilus JWO13 45-50 (~50) 2.0-4.0 (2.0-3.0) 2.1 5.20 x 105 (2/50)

5 S. acidophilus JWO16m 45-60 (~50) 2.0-5.0 (~2.0) 3.6 6.30 x 10 (2/50)

6 S. acidophilus JWO17m 45-50 (~50) 2.0-3.0 (~2.0) 2.6 7.55 x 10 (2/50)

6 S. acidophilus JWO18m 45-50 (~50) 2.0-3.0 (~3.0) 3.2 1.20 x 10 (3/50)

6 S. acidophilus JWO19m 45-50 (~50) 2.0-3.0 (~2.0) 3.1 4.63 x 10 (2/50)

6 S. acidophilus JWO20m 45-60 (~50) 2.0-3.0 (~2.0) 2.6 1.45 x 10 (2/60)

6 S. acidophilus JWO21m 45-50 (~50) 1.0-4.0 (~2.0) 3.2 8.08 x 10 (2/50)

5 S. acidophilus JWO22m 35-50 (~50) 2.0-5.0 (2.0-3.0) 4.4 9.45 x 10 (2/50)

Alicyclobacillus tolerans JAW5 45-50 (~50) 3.0 1.4 2.46 x 107 (3/50)

Alicyclobacillus sp. JWW6 45-60 (~50)` 3.0-5.0 (3.0-4.0) 0.8 8.44 x 107 (4/50)

Alicyclobacillus tolerans JAW7 30-50 (~50) 2.0-4.0 (~4.0) 2.2 7.00 x 107 (4/50)

Alicyclobacillus tolerans JAW8 30-50 (~50) 3.0-4.0 (~3.0) 1.9 5.25 x 107 (3/50)

Linear regression modeling of pH and temperature data. After graphing the

division rate (generations/time) (z) for each growth experiment, it was clear that the maximum rate for most of the Sulfobacillus strains occurred at 50˚C and pH 2.0, and

response values decreased in both the temperature (x) and pH (y) directions. This

curvature could only be described using higher order polynomial terms. A new data set

was constructed that included generations/time, pH, and temperature, as well as the actual

values of pH and temperature when raised to the 2nd, 3rd and 4th powers (i.e. x, x2, x 3, x

4, y, y2,y3,y4). While these exponentiated values contain little interpretable biological

information, they are important in terms of providing the numerical information

necessary for fitting the models.

To determine whether strain is an important predictor of growth rate, only utilizing the observations for Sulfobacillus, we tested 47 different linear models with

every different combination of predictors (x, x2, x 3, x 4, y, y2, y3, y4, and strain). While we

tested for interaction between ‘strain’ and each x and y (at each power), the lack of full replication prevented testing for interaction between temperature and pH. Experimental treatments were limited and did not include all possible temperatures and pH values.

Treatments only included a temperature range of 30, 35, 40, 50, 60, 70°C at a constant pH of 2.0 and a pH range of 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 at a constant temperature of

50°C. AICc scores pointed to 4 best-fit models that included 99% of the cumulative weight (Table 4), and only differed by whether to include a third or fourth order ‘x’ or ‘y’ variable. Neither of these included strain as a predictor. Diagnostics on the top model were checked by residual plots, and they were generally all agreeable, with the exception

of one observation (growth at pH 1.0) that was exerting extreme leverage. The R2 for the top model was 0.6219, indicating a reasonably good fit, given the number of parameters included in the model.

The addition of strain to a model added at least 11 parameters and several more when accounting for interactions of strain and pH/temperature. AIC penalizes for the addition of parameters, thus inclusion of strain may have been penalized in the AICc score. We attempted to reduce the number of parameters by grouping the strains into six physiological similar groups (Table 5). We then retested all the models with ‘strain’ as

well as ‘group’ replacing strain. After ranking the models, the top two were the same as

before, while the third model did contain group as a single categorical predictor (Table

6). However, the inclusion of group only raised the log-likelihood value from 91.017 to

91.0374 and increased the delta AICc by close to 2, indicating that ‘group’ was an uninformative pseudo-variable. Therefore, only pH and temperature were indicated as variables most likely to affect growth rate of Sulfobacillus strains. With these data we cannot distinguish different permissible growth ranges for pH and temperature among our

Sulfobacillus isolates.

When we ranked models for growth data of Acidimicrobium strains (including

ICPT) in addition to the Sulfobacillus strain data discussed above, the top 16 models

included species as a predictor with 100% cumulative weight (Table 7).

Table 4. AICc of the top linear regression models of division rate (generations/time) according to pH, temperature, and strain of Sulfobacillus isolates. Parameters include the average growth rate of duplicates incubated at 50˚C with varying pH (1.0- 6.0) and incubated across a temperature range of 30-60˚C at pH 2.0. Data for Sulfobacillus isolates JAO1, JAO2, JWO12, JWO13, JWO16m, JWO17m, JWO18m, JWO19m, JWO20m, JWO22m were used in this study. Variables ‘x’ and ‘y’ refer to temperature and pH values tested for division rate ‘z’. Model* No. of Log- AICc Delta Akaike Cumulative parameters likelihood AICc weight weight lm(z~x+x2+x3+x4+y+y2+y3,data=polymic) 9 91.017 -160.983 0 0.536 0.536 lm(z~x+x2+x3+x4+y+y2+y3+y4,data=polymic) 10 91.7117 -159.63 1.3528 0.2725 0.8086 lm(z~x+x2+x3+y+y2+y3,data=polymic) 8 88.164 -157.928 3.0552 0.1163 0.9249 lm(z~x+x2+x3+y+y2+y3+y4,data=polymic) 9 89.0502 -157.05 3.9337 0.075 0.9999 lm(z~x+x2+x3+y+y2,data=polymic) 7 79.2242 -142.612 18.3708 0.0001 1 lm(z~x+x2+x3+y,data=polymic) 6 77.1392 -140.924 20.0597 0 1 lm(z~x+x2+x3+x4+y+y2+y3+strain,data=polymic) 19 96.4441 -139.378 21.6052 0 1

Table 5. Sulfobacillus isolate sub-groups organized by common temperature and pH ranges/optima. All isolates were grown in Heterotrophic medium held at pH 2.0 for temperature experiments and incubated at 50˚C for pH experiments. Group Sulfobacillus sp. isolates pH range (optimum) Temperature (˚C) 1 JAO1, JWO16m 2-5 (2-3) 45-60; opt. ~50 2 JAO2, JWO12, JWO13 2-4 (2-3) 45-50; opt. ~50 3 JWO17m, JWO18m, JWO19m 2-3 (2) 45-50; opt. ~50 4 JWO20m 2-3 (2) 45-60; opt. ~50 5 JWO21m 1-4 (2) 45-50; opt. ~50 6 JWO22m 2-5 (2-3) 35-50; opt. 50

Table 6. AICc of the top linear regression models of growth rate (generations/time) according to pH, temperature, strain, and/or sub-groups of Sulfobacillus sp. isolates. Parameters include the average growth rate of duplicates incubated at 50˚C with varying pH (1.0-6.0) and incubated across a temperature range of 30-60˚C at pH 2.0. Data for Sulfobacillus isolates JAO1, JAO2, JWO12, JWO13, JWO16m, JWO17m, JWO18m, JWO19m, JWO20m, JWO22m were used in this study. ‘Group’ refers to 6 sub-groups of common physiology from the 10 isolates tested (Table 5). Variables ‘x’ and ‘y’ refer to temperature and pH values tested for growth rate ‘z’. Model* No. of Log- AICc Delta Akaike Cumulative parameters likelihood AICc weight weight lm(z~x+x2+x3+x4+y+y2+y3,data=polymic11) 9 91.017 -160.983 0 0.2945 0.2945 lm(z~x+x2+x3+x4+y+y2+y3+y4,data=polymic11) 10 91.7117 -159.63 1.3528 0.1498 0.4443 lm(z~x+x2+x3+x4+y+y2+y3+group,data=polymic11) 10 91.0374 -158.282 2.7015 0.0763 0.5206 lm(z~x+x2+x3+y+y2+y3,data=polymic11) 8 88.164 -157.928 3.0552 0.0639 0.5845 lm(z~x+x2+x3+y+y2+y3+group+group:x,data=polymic11) 10 90.8571 -157.921 3.0622 0.0637 0.6482 lm(z~x+x2+x3+y+y2+y3+group+group:x+group:y+group:x2,data= 12 93.6861 -157.801 3.1825 0.0600 0.7082 polymic11) 67

Table 7. AICc of the linear regression models of growth rate (generations/time) according to pH, temperature, and species of Acidimicrobium and Sulfobacillus. Parameters include the average growth rate of duplicates incubated at 50˚C with varying pH (1.0-6.0) and incubated across a temperature range of 30-60˚C at pH 2.0. Species of Sulfobacillus included isolates JAO1, JAO2, JWO12, JWO13, JWO16m, JWO17m, JWO18m, JWO19m, JWO20m, and JWO22m. ‘Species’ of Acidimicrobium included EAO1, EAO2, EAO4, and A. ferrooxidans ICPT. Variables ‘x’ and ‘y’ refer to temperature and pH values tested. No. of Log- Delta Akaike Cumulative Model Equation AICc parameters likelihood AICc weight weight lm(z~x+x2+x3+x4+y+y2+y3+y4+species+species:y+species:x,data=poly 13 125.8514 -220.913 0 0.3864 0.3864 mic11) lm(z~x+x2+x3+x4+y+y2+y3+y4+species+species:y+species:x+species:x 14 126.327 -219.054 1.8593 0.1525 0.539 2,data=polymic11) lm(z~x+x2+x3+y+y2+y3+species+species:x+species:y,data=polymic11) 11 121.8007 -218.217 2.6964 0.1004 0.6393 lm(z~x+x2+x3+x4+y+y2+y3+species,data=polymic11) 10 120.2585 -217.732 3.1811 0.0788 0.7181 lm(z~x+x2+x3+x4+y+y2+y3+y4+species+species:x,data=polymic11) 12 122.3777 -216.703 4.2099 0.0471 0.7652 lm(z~x+x2+x3+x4+y+y2+y3+y4+species+species:y+species:x+species:x 15 126.3956 -216.305 4.6086 0.0386 0.8038 2+species:y2,data=polymic11) lm(z~x+x2+x3+y+y2+y3+species+species:x+species:y+species:x2,data= 12 122.1433 -216.235 4.6788 0.0372 0.841 polymic11) lm(z~x+x2+x3+y+y2+y3+species+species:x+species:y+species:y2,data= 12 121.8452 -215.638 5.275 0.0276 0.8687 polymic11) lm(z~x+x2+x3+x4+y+y2+y3+y4+species+species:y,data=polymic11) 12 121.7834 -215.515 5.3985 0.026 0.8947 lm(z~x+x2+x3+x4+y+y2+y3+y4+species,data=polymic11) 11 120.402 -215.419 5.4939 0.0248 0.9194 lm(z~x+x2+x3+x4+y+y2+y3+y4+species,data=polymic11) 11 120.402 -215.419 5.4939 0.0248 0.9442 lm(z~x+x2+x3+y+y2+y3+species+species:x,data=polymic11) 10 118.7697 -214.755 6.1586 0.0178 0.962 lm(z~x+x2+x3+y+y2+y3+species+species:y,data=polymic11) 10 118.6017 -214.419 6.4946 0.015 0.977 lm(z~x+x2+x3+y+y2+y3+species,data=polymic11) 9 117.2926 -214.335 6.5781 0.0144 0.9914 lm(z~x+x2+x3+y+y2+y3+y4+species,data=polymic11) 10 117.5678 -212.351 8.5626 0.0053 0.9968 lm(z~x+x2+x3+y+y2+y3+species+species:x+species:y+species:y2+speci 15 123.914 -211.342 9.5718 0.0032 1.0000 es:x2+species:y3+species:x3,data=polymic11)

Evaluation of iron and pyrite as potential energy sources

For A. ferrooxidans ICPT, growth rates and max cell densities in Autotrophic medium increased with increasing iron concentrations (up to 50 mM) when grown with a filter-sterilized air headspace (Fig. 15a). Cultures grown in 50 mM FeSO4 with a CO2

enhanced headspace had a slightly higher growth rate and yield when compared to

growth in the lower iron concentrations (Fig. 15b), but differences in yield and rates were

less pronounced (than for air-grown cultures), and the results for growth in 25 mM FeSO4

were somewhat anomalous with apparent yield decreasing after 48 hrs. Iron

concentrations ≤10mM had similar growth rates/max yields whether grown in headspace

of enhanced CO2 or air.

Conversely, EAO1 and EAO4 isolates cultured in Autotrophic medium with

5 either air- or CO2- enhanced atmosphere rarely reached 10 cells/ml (Fig. 15c, d) (EAO2

not tested). Moreover, similar or greater yields were obtained in the inoculated negative

control (containing no iron) for both EAO1 and EAO4 air-cultures and EAO4 CO2-

cultures than in tubes with iron. No growth was observed for the inoculated (no iron)

control for EAO1 CO2-cultures (not shown). Therefore, autotrophic growth of EAO1 and

EAO4 was not observed for cultures regardless of the headspace gas composition, or iron

concentration.

All Acidimicrobium strains examined in Mixotrophic media (i.e. containing yeast

extract) had significantly greater growth rates and reached a maximum density of at least

two orders of magnitude higher than was observed in Autotrophic media (Compare Figs.

15 and 16). To determine the minimum concentration of YE required for optimal growth,

we compared growth of EAO1 in Mixotrophic medium with varying YE concentrations

of 0%, 0.01%, 0.02%, and 0.025% (not shown). There was no difference between growth

rates and maximum cell yields for 0.01%-0.025% YE. Therefore we used the lowest

yeast extract (0.01%) concentration conducive for growth for all other mixotrophic

studies.

The highest yields (~107cells/ml) of A. ferrooxidans type strain ICPT were

observed in Mixotrophic and Heterotrophic medium (Fig. 16a). Collectively, cultures

grown in 1-50 mM FeSO4 Mixotrophic medium had faster doubling times (~4.4 hrs) than when grown in the same medium lacking iron (doubling time 12.5 hrs). The type strain

5 had minimum to no growth (yields ≤1x10 cells/ml) in Pyrite/YE medium, Mixotrophic

media without YE, or the negative control without YE or iron.

EAO1 grew the fastest (doubling time of 4.2 hrs) and reached the highest cell

7 densities (~10 cells/ml) when grown with 1-10 mM FeSO4 in Mixotrophic and

Heterotrophic medium (Fig. 16b). The next fastest doubling times were ~4.8hrs, 5.3hrs, and 5.7hrs for 25-50 mM Mixotrophic, Mixotrophic without iron media, and 1%

Pyrite/YE medium, respectively. Growth in all three media reached similar yields of

5x106 cells/ml. Minimal to no growth (yields ≤1x105 cells/ml) was observed in

Mixotrophic medium without YE, or the negative control without YE and iron. Optimal

growth of EAO isolates in terms of yield and doubling time occurred in Mixotrophic

media at 0.036-10 mM FeSO4/0.01%YE.

The fastest doubling time (3.2 hrs) and yields (6x106 cells/ml) for Sulfobacillus

JWO19m were observed in Heterotrophic and 1 mM Mixotrophic media (Fig. 16c). On

Pyrite, maximum yields were also 6x106 cells/ml, but doubling times were approximately

twice as long (5.8 hrs) when compared to growth in Heterotrophic or 1 mM FeSO4

Mixotrophic media. Similar doubling times (3.4-3.9 hrs) and yields (2-4x106 cells/ml)

were observed in 10-50 mM FeSO4 Mixotrophic, and YE with no iron. Minimal to no growth (yields ≤1x105 cells/ml) was observed in Mixotrophic medium without YE, and

the negative control without YE or iron.

In summary, autotrophic growth was only evident for ICPT type strain at 25-50 mM iron grown with filter sterilized air (Fig. 15). EAO isolates had minimal to no growth under autotrophic conditions. All Acidimicrobium and Sulfobacillus strains tested had significantly shorter doubling times and higher maximum cell yields in Mixotrophic/

Heterotrophic media versus Autotrophic, indicating preferred growth in the presence of yeast extract (Fig. 16). Mixotrophic growth of EAO isolates was optimal for iron concentration 1-10 mM suggesting inhibition at higher iron concentrations (≥25mM).

EAO and Sulfobacillus isolates examined were able to grow in 1% Pyrite medium in the presence of YE, however A. ferrooxidans ICPT did not.

T T a) A. ferrooxidans ICP w/ air b) A. ferrooxidans ICP w/ CO2 1.00E+06 50 mM Fe 1.00E+06 25 mM Fe 10 mM Fe

0.68 M Fe

1.00E+05 1.00E+05 0.136 mM Fe

cells / ml cells

cells / ml

1.00E+04 1.00E+04

1.00E+03 1.00E+03 0 20 40 60 80 100 120 0 20 40 60 80 100 120 time / (hr)hr timetime / (hr)hr c) EAO4 w/ air d) EAO4 w/ CO2 50 mM Fe 25 mM Fe 1.00E+05 1.00E+05 10 mM Fe 2 mM Fe 0.68 mM Fe 0.136 mM Fe

negative control

cells / ml 1.00E+04 cells / ml cells 1.00E+04

1.00E+03 0 10 20 30 40 50 60 70 80 1.00E+03 timetime / hr(hr) 0 10 20 30 40 50 60 70 80 timetime / hr(hr)

Figure 15. Comparison of autotrophic growth at varying iron concentrations, T supplemented with either air or enhanced CO2, between A. ferrooxidans ICP (top) and isolate EAO4 (bottom). Duplicates were grown in Autotrophic medium at varying iron concentrations (mM Fe) and incubated at 45°C. Culture headspaces were replenished with either filter-sterilized air (left) or enhanced CO2 (3-5% v/v) (right) after inoculation and before incubation. Only EAO isolates included a negative control of cultures inoculated into medium with no iron (basal salts only). EAO1 (not shown) had similar results as EAO4. Error bars represent standard error.

T a) A. ferrooxidans ICP b) Acidimicrobium EAO1 c) Sulfobacillus JWO19m 50 mM Fe/YE 1.0E+07 1.0E+07 1.0E+07 25 mM Fe/YE 10 mM Fe/YE 1 mM Fe/YE 1% pyrite/YE 1.0E+06 hetero 1.0E+06 1.0E+06

YE only

10 mM Fe only

negative control

1.0E+05 1.0E+05 1.0E+05 cells / ml cells cells / ml cells / ml cells

1.0E+04 1.0E+04 1.0E+04

1.0E+03 1.0E+03 1.0E+03 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 time / hr time (hr) timetime (hr)/ hr timetime /(hr) hr

Figure 16. Mixotrophic growth comparison of A. ferrooxidans ICPT (a), Acidimicrobium EAO1 (b), and Sulfobacillus JWO19m (c). Duplicates of each organism were inoculated into the following pH 2.0 medium and incubated at 50°C: Mixotrophic at varying iron concentrations (Fe/YE), 1% Pyrite/YE, Heterotrophic (hetero) adjusted to 0.01% YE, Mixotrophic without iron (YE only), Autotrophic adjusted to 10 mM FeSO4 (10 mM Fe only), and Mixotrophic without iron or YE as the negative control. Error bars are standard error.

Measurements of iron oxidation

As cultures of Acidimicrobium ferrooxidans ICPT and Acidimicrobium EAO

isolates reached their maximum cell densities, all organisms oxidized ferrous iron

completely in 10 mM FeSO4 Mixotrophic medium (Fig. 17). The minimum time required

for complete oxidation varied among Acidimicrobium strains; it took 48 hrs for the ICPT

type strain (Fig. 17a), 72 hrs for EAO1 (Fig. 17b), 120 hrs for EAO2 (Fig. 17c), and 140 hrs for EAO4 (not shown). Doubling times and maximum yields of the type strain were comparable to previous growth on Mixotrophic medium (Fig. 16). Results for mixotrophic growth of EAO1 were slightly less (doubling time: 5.7 hrs; yield: 1.12x106

cells/ml) than was previously observed (Fig. 16). Doubling times and maximum cell

yields for EAO2 and EAO4 were similar; 7.0-8.4 hrs and 3-5x105 cells/ml, respectively.

Isolates EAO2 and EAO4 showed oxidation of iron in YE-free- Mixotrophic medium

even though they showed minimal growth, only reaching densities of 1x105 cells/ml. In

the absence of YE, EAO2 oxidized ~8mM ferrous iron in 120 hrs. EAO4 oxidized ~6

mM in 120 hrs. EAO1 showed some iron oxidation (~3 mM in 90 hrs), however slight

abiotic oxidation in glutaraldehyde-killed controls was also ~3mM). A. ferrooxidans

ICPT did not show any evidence of iron oxidation without YE even though minimal

growth was observed (Fig. 17a). Slight abiotic oxidation was seen in glutaraldehyde-

killed controls, but not in uninoculated controls.

In the presence of 0.01% YE, all Sulfobacillus isolates tested were able to oxidize

10 mM ferrous iron to 2 mM or less (duration: 40-50 hours) (represented by JAO1, Fig.

17d). Ferrous iron was oxidized by the majority of Sulfobacillus isolates to ~6 mM in

cultures without YE even though no growth was observed. Only JWO19m showed

evidence of some growth in the absence of YE by reaching cell densities slightly above

1x105 cells/ml and oxidized ~7 mM ferrous iron (not shown). Maximum cell densities of

JWO19m were similar to the growth observed in Mixotrophic medium (Fig. 16c). Slight

abiotic iron oxidation was seen in glutaraldehyde-killed controls, but not in uninoculated

controls.

A summary for iron oxidation studies for all isolates tested is found in Table 8.

Pyrite oxidation

At time zero, ~3.5 mM ferrous iron was already present in uninoculated 1% Pyrite medium (Fig. 18). In the presence of 0.01% YE, all EAO isolates were able to oxidize this ferrous iron completely by 50-90 hours post inoculation (Fig. 18a). EAO2 and EAO4 had similar faster doubling times (4.8-5.1 hrs) and greater yields (2x107 cells/ml) than

EAO1 (~6 hrs and 5x106 cells/ml) (not shown). Growth rates and maximum cell yields of

EAO1 were similar to growth observed in Pyrite/YE medium (compare Figs. 16b and

18a). Slight oxidation (1-1.5 mM) of ferrous iron was observed in EAO cultures grown

in Pyrite without YE. Maximum cell yields for all EAO isolates in Pyrite without YE was

1x105 cells/ml. A. ferrooxidans ICPT did not grow in Pyrite medium (with or without

yeast) nor did it oxidize iron present in the medium (data not shown).

EAO isolates in Pyrite/YE medium were followed for evidence of pyrite

oxidation by monitoring pyrite dissolution (increase in total soluble iron). At time zero,

there was ~5 mM total soluble iron already present in the Pyrite medium at pH 2.0 (Fig.

19); ~3.5 mM of the soluble iron is present as ferrous iron (Fig. 18) and ~1.5 mM is present as ferric iron. No dissolution of ground rock pyrite was observed over the 20 day incubation period for either EAO isolate (Fig. 19a). Initial pH of cultures in Pyrite medium was ~2.01. At the end of the 20 day incubation period, the pH decreased in all cultures from 1.75-1.81. Abiotic dissolution of pyrite and pH changes were not observed in negative controls over the 20 day incubation.

Of the six Sulfobacillus isolates investigated, only JWO19m (Fig. 18b) and

JWO22m (not shown) were able to grow and oxidize iron present in Pyrite medium amended with 0.01%YE. Doubling times and yields of JWO19m were similar to growth in Pyrite/YE medium (Compare Figs. 16c and 18b). Isolate JWO19m was also able to grow in Pyrite medium without YE (yield ~2x105 cells/ml) and oxidized ~2m M Fe2+ present in Pyrite medium (Fig. 18b).

Isolates JWO19m and JWO22m were followed for 20 days to determine if they were capable of pyrite dissolution. In 18 days, isolates JWO19m and JWO22m increased total soluble iron by 3.5 mM and 2 mM, respectively (Fig. 19b). The average pH observed after incubation for JWO19m and JWO22m was 1.70 (a change of -0.30) and

1.74 (a change of -0.26), respectively. No abiotic dissolution or changes in pH were observed in negative controls over the 20 day incubation period. A summary for pyrite oxidation studies for all isolates tested can found in Table 8.

77 T a) A. ferrooxidans ICP b) Acidimicrobium EAO1 1.0E+07 12 1.0E+07 12 Fe/YE growth Fe growth

glut-killed growth 10 10

Fe/YE mM [Fe II] 1.0E+06 1.0E+06

Fe mM [Fe II] 8 8

glut-killed mM [Fe II] uninoculated mM [Fe II] 1.0E+05 6 1.0E+05 6 cells / ml cells cells / ml mM [FeII] mM mM [Fe II]

4 4

1.0E+04 1.0E+04

2 2

1.0E+03 0 1.0E+03 0 0 10 20 30 40 50 0 10 20 30 40 50 60 70 80 timetime/hr (hr) timetime/hr (hr) c) Acidimicrobium EAO2 d) Sulfobacillus JAO1 1.0E+07 12 1.00E+07 12

10 10

1.0E+06 1.00E+06

1.0E+05 6 1.00E+05 6 cells / ml cells / ml mM mM [Fe II] mM mM [Fe II]

4 4

1.0E+04 1.00E+04

2 2

1.0E+03 0 1.00E+03 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 time/hr timetime/hr (hr) time (hr) Figure 17. Comparison of growth and concomitant iron oxidation by Acidimicrobium and Sulfobacillus strains. The average of duplicate samples was plotted over time for A. ferrooxidans ICPT (a), Acidimicrobium EAO1 (b), Acidimicrobium EAO2 (c), and Sulfobacillus JAO1 (d). Acidimicrobium EAO4 had similar results as EAO2, and all Sulfobacillus isolates (with the exception of JWO19m) had similar results as JAO1. Double-Y axis graph shows growth (cells/ml) (left) and concentration of ferrous iron (mM [Fe II]) (right) over time in Mixotrophic medium with 0.01%YE (circle) and without YE (triangle), incubated at 50°C. Growth curves are represented by solid lines and ferrous iron oxidation is represented by dotted lines. Negative controls were glutaraldehyde-killed EAO1/JAO1 (square), and uninoculated Mixotrophic medium (black dashed line). Glutaraldehyde-killed cells were fixed near potential maximum cell densities of ~106 cells/ml for Acidimicrobium (EAO1; a, b, c) and Sulfobacillus (JAO1; d). Regardless of medium type, negative controls had similar results. Slight apparent abiotic oxidation of iron was seen in killed controls but not in uninoculated controls. Error bars represent standard error.

a) Acidimicrobium EAO4 1.00E+07 b) Sulfobacillus JWO19m P/YE growth 1.00E+07 14 14 P growth

glut-killed growth 12 12

P/YE mM [Fe II] 1.00E+06

P mM [Fe II] 1.00E+06 10 10

glut-killed mM [Fe II]

uninoculated mM [Fe II] 8 8 1.00E+05

1.00E+05 cells / ml cells mM [Fe II] 6 6 cells / ml cells 4 4 mM [Fe II] 1.00E+04 1.00E+04

2 2

1.00E+03 0 1.00E+03 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 timetime /(hr) hr time (hr)/ hr

Figure 18. Comparison of iron oxidation in 1% Pyrite medium by Acidimicrobium EAO4 (a) and Sulfobacillus JWO19m (b). Double-Y axis graph shows growth (cells/ml) (left) and concentration of ferrous iron (mM [Fe II]) (right) over time. Duplicates were grown in 1% Pyrite medium with 0.01%YE (circle) and without 0.01%YE (triangle) and incubated at 50˚C. Growth curves are represented by solid lines, and ferrous iron oxidation is represented by dotted lines. Negative controls were glutaraldehyde-killed EAO1 (a) or JAO1 (b) (square) and uninoculated (black dashed line) in Mixotrophic medium. Glutaraldehyde-killed controls were inoculated at near maximum cell densities. All Acidimicrobium isolates tested have similar growth and iron oxidation results as seen in (a). Only JWO19m and JWO22m were able to grow on 1% Pyrite with 0.01%YE; no other Sulfobacillus isolate grew in either Pyrite medium type. Standard error bars are shown.

a) Acidimicrobium isolates b) Sulfobacillus JWO19m and JWO20m 1.00E+08 1.00E+07 EAO1 growth JWO19m growth 14.00 14 EAO2 growth JWO22m growth EAO4 growth 12.00 12 glut-killed growth 1.00E+07 glut-killed growth 1.00E+06 EAO1 mM [Fe] JWO19m mM [Fe] 10.00 10 EAO2 mM [Fe]

JWO22m mM [Fe]

EAO4 mM [Fe] 8.00 8 glut-killed mM [Fe] 1.00E+06 glut-killed mM [Fe] 1.00E+05 cells / ml cells cells / ml uninoculated mM [Fe] uninoculated mM [Fe] 6.00 6 mM [Total Soluble Fe] [Total mM mM [Total Soluble Fe] mM [Total 4 1.00E+05 4.00 1.00E+04

2.00 2

1.00E+03 0 1.00E+04 0.00 0 5 10 15 20 25 0 5 10 15 20 25 timetime /(day) day timetime (day)/ day Figure 19. Comparison of dissolution of pyrite by Acidimicrobium (a) and Sulfobacillus (b) isolates. Double-Y axis graph showing growth (cells/ml) (left) and concentrations of total soluble iron (mM [Fe]) (right) over ~20 days. Duplicates were grown in 1% Pyrite medium with 0.01%YE, incubated at 50˚C, and refreshed with filter-sterilized air every 5 days. Growth curves are represented by solid lines and ferrous iron oxidized is represented by dotted lines. Negative controls were glutaraldehyde-killed EAO1 (a) or JAO1 (b) (square) and uninoculated (black dashed line) in Mixotrophic medium. Glutaraldehyde-killed controls were inoculated at near maximum cell densities. After 2-3 days, all isolates reached maximum cell densities and oxidized ferrous iron present (~3.5 mM) in pyrite medium. During this time, no change in total soluble iron was observed. However, only Sulfobacillus isolates JWO19m and JWO22m showed an increase in total soluble iron suggestive of pyrite oxidation. Standard error bars are shown.

Sulfur and tetrathionate oxidation

In the presence and absence of trace iron (0.036 mM), growth of A. ferrooxidans

ICPT and EAO isolates in Sulfur/YE and Sulfur/YE/trace Fe media resulted in similar average cell yields ~1x107 cells/ml (Fig. 20). However, there was an initial lag phase of

~22 hr and ~45 hr for EAO cultures in Sulfur/YE and Sulfur/YE/trace Fe, respectively.

No growth was observed for either Acidimicrobium strain in Sulfur medium without YE

(not shown).

There was no difference in doubling time or maximum yields for either strain

between Heterotrophic and Heterotrophic/0.01% YE media. These media only differ in

terms of YE concentrations: Heterotrophic contains 0.025% and Heterotrophic/0.01% YE

contains 0.01%. Strain ICPT had slightly faster doubling times and greater growth yields

in Heterotrophic and Heterotrophic/0.01%YE media than S/YE, S/YE/trace Fe and YE

media (Fig 20a). This suggests that the addition of trace iron did not affect growth of the

type strain in the presence of sulfur. Even though EAO strains had an initial lag phase for growth in sulfur-containing media, they reached 2X higher cell densities in media containing sulfur than without (Fig. 20b). The lowest yields for EAO strains occurred in

YE medium at 3x106 cells/ml suggesting that sulfur or trace iron positively affect growth.

The pH of all the cultures reduced slightly over time by ~0.05, however, there was no

difference between pH changes observed in inoculated versus uninoculated tubes.

No growth was observed in Tetrathionate media, with or without YE, for any of

the Acidimicrobium strains (Fig. 20). Moreover, there was no change in culture pH over

the 100-140 day incubation period.

All Sulfobacillus isolates had similar growth yields (1-3x106 cells/ml) in

Sulfur/YE media (Fig. 21). Collectively, growth yields in Sulfur media without YE were

very low and ranged from ~5x104-1x105 cells/ml. There was no difference between

maximum yields of isolates JAO1, JWO20m, JWO21m, and JWO22m in Sulfur/YE versus

YE media (Fig. 22a). Only JWO19m had slightly greater yields in Sulfur/YE medium

(3x106 cells/ml) versus YE medium (1x106 cells/ml) (Fig. 21b). There was no change in

culture pH over the 100 day incubation period for any of the isolates tested.

Sulfobacillus isolates JAO1, JWO19 m, JWO20 m, JWO21 m, and JWO22 m did

grow in Tetrathionate/YE (Tet/YE) medium with yields ranging from 5x106-9x106

cells/ml (Fig. 21). With the exception of JAO1, all other Sulfobacillus isolates had

doubling times ~2X slower in Tet/YE versus YE media (Fig. 21b). Since both medium

types contained YE but differed with the presence or absence of tetrathionate, growth of

these cultures was inhibited by tetrathionate. Only JAO1 had similar growth rates and

yields in Tet/YE and YE media (Fig. 21a).

Three isolates JAO1, JWO19m, and JWO22m, were also inoculated into a lower

concentration of tetrathionate (2.5 mM) to see if tetrathionate-mediated growth inhibition occurred. The two isolates (JWO19m and JWO22m) whose growth was inhibited in

Tet/YE media containing 5 mM Tetrathionate were not inhibited in 2.5 mM Tet/YE

media (Fig. 21b); growth rates and yields in 2.5 mM Tet/YE were similar to YE medium.

For JAO1, no growth was observed in 2.5 mM Tet/YE.

No growth was observed for the majority of Sulfobacillus isolates in Tetrathionate

medium (without YE) at either tetrathionate concentration (Fig. 21). Again, only JAO1

82 showed evidence of growth in 5 mM Tetrathionate medium (without YE), with yields of

3x105 cells/ml (Fig. 21a). There was no change in culture pH of Tetrathionate media

(with or without YE) over the 100 day incubation period for any of the isolates.

A summary of growth in sulfur and tetrathionate results for all genera tested are found in Table 8.

a) A. ferrooxidans ICPT b) Acidimicrobium EAO4 1.0E+08 1.00E+08

1.0E+07 1.00E+07

1.0E+06 1.00E+06 S/YE S/YE S/YE/trace Fe S/YE/trace Fe Hetero/0.01% YE cells / ml Hetero/0.01% YE cells / ml Hetero 1.0E+05 Hetero 1.00E+05 YE YE T/YE

1.0E+04 1.00E+04

1.0E+03 1.00E+03 0 20 40 60 80 100 120 0 20 40 60 80 100 120 140 160 timetime / hr(hr) timetime (hr)/ hr

Figure 20. Comparison of growth in Sulfur and Tetrathionate media by A. ferrooxidans ICPT (a) and Acidimicrobium isolate EAO4 (b). Duplicates were incubated at 50˚C and inoculated into the following media: Sulfur with 0.01%YE (S/YE), Sulfur with 0.01%YE and 0.036 mM FeSO4 (S/YE/trace Fe), Tetrathionate with 0.01%YE (T/YE), Heterotrophic (Hetero), Heterotrophic adjusted to 0.01%YE (Het/0.01%YE), and YE. Cultures that exceeded 5 day incubation were refreshed with filter-sterilized air every 5 days. The averages of duplicates are plotted with error bars representing standard deviation. Results for all Acidimicrobium isolates (not shown) were similar to EAO4. The type strain was unable to grow on tetrathionate with or without YE (data not shown).

a) Sulfobacillus JAO1 b) Sulfobacillus JWO19m 1.00E+07 S/YE 1.00E+07 S 5 mM T/YE 5 mM T 1.00E+06 2.5 mM T/YE 1.00E+06 2.5 mM T YE

1.00E+05 1.00E+05 cells / ml cells cells / ml cells

1.00E+04 1.00E+04

1.00E+03 1.00E+03 0 20 40 60 80 100 0 20 40 60 80 timetime /(hr) hr timetime (hr)/ hr

Figure 21. Comparison of growth in Sulfur and Tetrathionate media by Sulfobacillus isolates JAO1 (a) and JWO19m (b). Duplicates were incubated at 50˚C and inoculated into the following media: Sulfur with 0.01%YE (S/YE), Sulfur (S), Tetrathionate (5 mM T), Tetrathionate with 0.01% YE (5 mM T/YE), Tetrathionate adjusted to 2.5 mM (2.5 mM T), Tetrathionate adjusted to 2.5 mM with 0.01% YE (2.5 mM T/YE), and YE. The averages of duplicates are plotted with error bars representing standard error. JAO1 was the only Sulfobacillus isolate able to grow in 5 mM tetrathionate. Results for all other Sulfobacillus isolates were most similar to JWO19m, with a few exceptions (see Results: Sulfur and Tetrathionate Oxidation section). No cells were observed for JWO19m in 2.5mM T medium.

Table 8. Summary of iron, pyrite, sulfur and tetrathionate oxidation results for Acidimicrobium and Sulfobacillus. Duplicate tubes, per organism, were inoculated into the following media: Autotrophic adjusted to 10mM FeSO4 (Auto), Mixotrophic (Mixo), 1%Pyrite/0.01%YE (P/YE), 0.5%Sulfur/0.01%YE (S/YE), and 5 mM Tetrathionate/0.01%YE (T/YE) and incubated at 50°C (see Methods). Positive (+) or negative (-) for growth/activity was indicated. Cultures that reached a maximum cell density of 105 cells/ml were referred to as minimal growth (m). Apparent inhibition (I) was also observed for 5 mM T/YE and positive growth at a lower concentration of 2.5 mM was indicated.

Growth in Growth and Growth and Pyrite/YE Growth Fe2+ oxidation Fe2+ oxidation Growth Organism and in 5 mM in Auto in Mixo in S/YE dissolution T/YE medium medium of pyrite A. ferrooxidans ICPT m*/- +/+ -/- + -

Acidimicrobium sp. EAO1 m/- +/+ +/+ + -

Acidimicrobium sp. EAO2 m/+ +/+ +/+ + -

Acidimicrobium sp. EAO4 m/+ +/+ +/+ + -

S. acidophilus JAO1 m/+ +/+ -/- + + (-2.5 mM)

S. acidophilus JWO13 m/+ +/+ -/- + ND m/+ +/+ S. acidophilus JWO19m +/+ + I (+2.5 mM) m/+ +/+ S. acidophilus JWO20m -/- + I m/+ +/+ S. acidophilus JWO21m -/- + I m/+ +/+ S. acidophilus JWO22m +/+ + I (+2.5 mM) *Autotrophic growth of the ICPT type strain was observed at iron concentrations >25 mM and minimal growth at ≤10 mM.

CHAPTER 4: DISCUSSION

Attempts to Isolate Acidimicrobium from BSL

The purpose of this thesis project was to isolate Acidimicrobium; during these

attempts, we also isolated numerous Sulfobacillus and Alicyclobacillus (Table 2). Both

genera are commonly found alongside Acidimicrobium and are routinely isolated together

(14, 25, 59, 81, 84). Although Sulfobacillus and Alicyclobacillus are rare members of the

BSL community based on molecular surveys (116), they are among the most common

heterotrophic isolates obtained at acidic pH (47). It is often the case that molecular

approaches identify the dominant members of the community while cultivation methods

capture the rare members (100). Similarities and key differences in physiology among

isolates help to explain why certain organisms were easier to cultivate than others.

Spread plating from dilutions of BSL water did not produce Acidimicrobium

isolates which leads me to believe that higher cell densities are required to produce colonies (i.e. the plating efficiency of this genus never reaches 100%). Sulfobacillus and

Alicyclobacillus strains have been isolated using the spread plate method (Table 2) (47).

Commonly, dilutions of samples are done to prevent overcrowding of cells on plates because some organisms do not form colonies at high densities or multiple species may grow together in a single colony leading to erroneous identification of isolates (69).

However, this does not appear to be the case for EAO isolates since the minimum threshold for growth was ~2x103cells/ml (not shown). Among bacterial populations, the

“Allee effect” is a common phenomenon in which there is a positive correlation between

87 growth rate and inoculum population density until a maximum threshold is reached that is unique for each species and set of conditions (2). Populations of bacteria can be induced to grow by quorum sensing, and this is likely to play a role in threshold densities permissive for growth of many species.

EAO strain growth on FeSo plates appeared as orange smears instead of single, isolated colonies (Fig. 6). Since single colonies are presumed to derive from a single cell, they are the target growth morphology when purifying organisms on solid media. In previous Acidimicrobium isolation attempts, these smears were discarded since they were not thought to be a pure culture (Patricia Siering, personal communication). Preference for single colonies that evinced iron oxidation was often followed, leading to isolation of

Sulfobacillus strains instead of Acidimicrobium. By utilizing a PCR screening method with Acidimicrobium-specific primers, we were finally able to focus on the most promising cultures and successfully isolate Acidimicrobium. It is not uncommon for strains that were previously unable to grow on solid media, to adapt and form colonies on agar after several attempts (22). For example, during the isolation of Acidimicrobium strains ICPT and Y0018, difficulty in acquiring single colonies was also observed (24,

57). Single colonies were finally obtained after several weeks of serial cultures in

Autotrophic and Mixotrophic medium for ICPT and Y0018, respectively.

Cultivation of acidophilic organisms onto solid media has long been problematic

(43). Even though the same acidophiles can grow in non-gelled media with similar or identical components as gelled-media, reproducible growth on gelled-media was often impossible. In particular, iron oxidizers such as Leptospirillum ferrooxidans were

categorized as incapable of growth on solid media until recent advances addressed the

problems inhibiting growth of acidophiles (43, 55, 56). Many gelling agents, including agar, are polysaccharides that hydrolyze in hot, acidic liquors. Although heat sterilization of the acidic components and the gelling agents are done separately to help minimize hydrolysis, hydrolysis of gelling agents continues under incubation temperatures for acidophilic thermophiles. The polysaccharide backbone structure for agar (and agarose) is similar, and is composed of galactose residues or charged groups of mostly pyruvic acid (54). Chemolithotrophic acidophiles are highly sensitive to these soluble organic acids present in the acidified gelling media, particularly pyruvic acid. At low pH, the organic acids are mainly undissociated lipophilic acids that can readily pass through cellular membranes. Once inside the circumneutral cytoplasm of acidophiles, the acids dissociate and acidify the cytoplasm.

In order to circumvent organic acid toxicity from the hydrolysis of agarose, we

used the overlay technique for isolation (55, 56). Although we achieved “growth” in the form of smears for EAO isolates, we were unable to obtain single colonies. Thus, assessing whether a culture is free of contamination is difficult. There are a few reasons

why organisms are unable to grow as single colonies. Although components of un-gelled

and gelled media are similar, it is difficult to control for water availability and gas

concentrations. Plates that are incubated at > 50˚C are placed in sealed plastic containers

in order to reduce moisture loss. However, water loss is apparent as volume of plate

reduces over time and accumulation of water on lid increases. The majority of plate growth is found underneath the surface of the FeSo (and FeSo) plates instead of on the

surface where the ferric iron oxides precipitate. Due to the growth-inhibiting

concentrations of iron in FeSo plates (25 mM), EAO strains may prefer regions of the

plate where iron concentrations (particularly ferric iron) are lowest. By reducing iron

concentrations in FeSo plates (to 1 mM and 10 mM), I have been able to obtain more

repreducible growth of EAO isolates (not shown). In addition, microaerobic conditions

produce more reliable growth of EAO strains. Perhaps microaerobic conditions help

reduce iron oxidation rates by EAOs, thereby allowing the isolates to accumulate on the

surface of plates before the iron oxide precipitates become inhibitory. Although high iron

concentrations are inhibitory, enough iron needs to be present for the orange phenotype to

be visible to aid in preliminary identification of iron oxidizers present in media. For

instance, plates containing 1 mM FeSO4 produce translucent/white smears/tiny colonies

(not shown). Further work will be needed to determine optimal conditions for the

formation of colonies. Purity of samples is currently determined by verifying

homogenous cell morphology in cultures with phase contrast microscopy.

Isolation and physiological characterization of Acidimicrobium

Attempting to isolate Acidimicrobium was a challenging prospect. Even though

Acidimicrobium has been identified molecularly in multiple warm, acidic, mineral sulfide-rich environments (9, 34, 42, 67, 93), only three strains have been isolated, including the type strain A. ferrooxidans ICPT. The ICPT type strain was isolated from a

Krísuvík geothermal area in Iceland (24), and a member of the same species, strain TH3,

was isolated from a copper mine leach dump in the U.S. (80). Acidimicrobium spp.

Y0018 was isolated from Frying Pan Hot Spring at the outer fringe of the Norris Geyser

area in Yellowstone National Park (57). Unfortunately, water chemistry data from isolation source sites are scarce. The only information provided was for strain Y0018 (57)

where the authors described all six sample sites as acidic (pH 2.7-3.7) and variable in

temperature (30-83˚C). The only specific reference to source conditions for strain Y0018

was that it was isolated from one of the “cooler sites” that was <70˚C.

Nevertheless, we obtained three isolates EAO1, EAO2, and EAO4 that share 99%

16S rRNA gene sequence identity with Acidimicrobium spp. Y0018, the A. ferrooxidans

type strain ICPT, and to each other (Table 3). These Acidimicrobium isolates also shared

99% 16S rRNA gene sequence identity with the Acidimicrobium phylotype previously

identified as abundant in BSL sites A and D SSU rRNA gene clone libraries (11, 117).

The most effective method for isolation of Acidimicrobium EAO strains was by

enriching at 45˚C in 1% pyrite or in Autotrophic medium, and sub-culturing from

enrichments onto FeSo (single layer or overlay) plates (Table 3). These isolation methods

are consistent with isolation of both Y0018 and ICPT strains that were obtained by

enriching site water samples in 1% pyrite at pH 2.0 (24, 57). The growth morphology of

EAO strains on FeSo plates complicated its isolation since the strains did not form

colonies.

EAO isolates were found to have a narrow permissible range of growth around

45-50˚C, whereas A. ferrooxidans ICPT had a wide range of growth from 30-50˚C (Table

4). Even though the enrichment and isolation temperature was 45˚C, the optimum temperature for growth of EAO was closer to 50˚C (Fig. 13, Table 3, 4). EAO isolates

had significant differences in growth rate between 45˚C and 50˚C. Doubling time at 50˚C

was generally 2-4X greater than at 45˚C. This is not surprising considering that the

optimum temperature for EAO isolates reflects the constant summer temperatures of BSL at site A (50-55˚C) (116). In addition, the lower isolation temperature may have

contributed to slowing growth rates of other organisms present in enrichments that may

have out-competed Acidimicrobium strains if grown at 50˚C. The A. ferrooxidans ICPT

strain was reported to have an optimum temperature of 48˚C (24), however I did not find

a significant difference between growth rates or maximum cell densities reached between

45˚C and 50˚C. This may explain why authors alternate between the two stated

temperature optima. Even though EAO isolates were unable to grow at 60˚C and above,

presence of Acidimicrobium 16S rRNA phylotypes from site D samples (~85˚C) can be

explained by the convective mixing of BSL (117).

However, considering the narrow permissible temperature range for growth of

EAO isolates, it is surprising that Acidimicrobium isolates are able to survive during

severe temperature drops in the winter. Winter temperatures of 37-45˚C have been

monitored at below the observed minimum for growth of EAO isolates (116, 117).

Acidimicrobium may not be actively growing at these temperatures, but this non-spore-

forming bacterium may be able to tolerate temperatures beyond its limits of growth. A

large number of non-spore-forming bacteria are capable of maintaining cellular structure

and continue significant gene expression under a dormancy state known as viable but

nonculturable (VBNC) (28, 85). The common response of VNBC cells to stress is the

inability to develop into colonies on routine culture media even though cells may remain

viable for long periods of time. Although much of the work has focused on Gram

negative bacteria and pathogens, many nonpathogenic and Gram positive bacteria are

capable of entering a VBNC state (85). Cells that enter a VBNC state are induced by a

number of chemical and environmental factors such as nutrient starvation (29) and

incubation outside the normal temperature range of growth (118). Cells undergo a reduction in size (dwarfing), reduction in nutrient transport, respiration rates, and macromolecular synthesis (85). During the period of VBNC in Vibrio sp., biosynthesis continues, ATP levels remain high and novel starvation and cold shock proteins are formed. In addition, extensive modifications to the cell walls of VBNC cells versus exponentially growing cells appear to be a hallmark for distinguishing a VBNC state

(104). Rearrangements of cell walls have been observed in both cells of VBNC Gram positive and Gram negative bacteria (30, 104). It has been suggested that dormancy and resuscitation from this state could indicate a strategy to test the suitability for growth of the environment (37). A few resuscitated cells may act as “scouts” and send a quorum sensing signal to the remaining VBNC cells indicating that the stressful environment is now permissive for growth. Overall, results for EAO isolates from temperature and pH growth studies strongly suggest that Acidimicrobium strains are active in BSL. In the future, we will need to test viability of EAO isolates or ability to enter a VBNC state after induction at temperatures outside their permissible range of growth.

Acidimicrobium EAO isolates were inhibited by iron concentrations ≥25 mM and grew optimally at 0.036-10 mM (Fig. 16b); this is surprising given the maximal growth

T of A. ferrooxidans ICP in 50 mM FeSO4. Yet, the lower acceptable range of iron for

EAO isolates is consistent with iron concentrations found at BSL (103). Although the iron concentration in BSL is considered high for a natural environment (~38 ppm; 0.68 mM) (102), the concentration of iron in isolation media Autotrophic (50 mM) and FeSo plates (25 mM ) was much higher in order to select for iron-oxidizers. Among iron-

oxidizing acidophiles, Sulfobacillus and others have a higher tolerance for the end

product of iron oxidation (i.e. Fe3+) than Acidimicrobium species (25). Researchers

initially pursued isolation of Acidimicrobium-like organisms in order to obtain more

efficient thermophilic, acidophilic iron-oxidizing bacteria for extraction of metals from materials containing high mineral sulfide concentrations (i.e. biomining) (16, 24, 81, 83).

Such an organism would also have to be capable of tolerating the harsh conditions in

biomining reactors (i.e. high concentration of metals) (24). Lower iron concentrations in

isolation media would select for a wider range of iron-oxidizing phenotypes. For this

reason, researchers may have intentionally selected an unnaturally high iron

concentration for isolation in order to select for strains that could be active members in

the biomining consortia. In order to obtain different species or ecotypes of

Acidimicrobium from BSL in the future, we will need to lower the iron concentrations in

isolation media.

Autotrophic growth using ferrous iron as an energy source was very poor for

EAO isolates, rarely reaching 1x105 cells/ml (Fig. 15c, d). However, unlike A.

T ferrooxidans ICP , EAO isolates did show evidence of iron oxidation at 10 mM FeSO4

(Fig. 18) even though cell yields were minimal. Maximum cell yields of the type strain

were 8X greater at 50 mM FeSO4 than 10 mM (Fig. 15a) in Autotrophic media. In our

study, we analyzed autotrophic iron oxidation for the type strain and our isolates at 10

mM FeSO4 whereas the previously reported studies of the type strain used 50 mM (24).

However, 50mM FeSO4 is inhibitory to EAO isolates. If this study were repeated, I

would expect to see evidence of iron oxidation for the type strain at 50 mM FeSO4 given

that cell yields were slightly higher at this iron concentration. Previous studies for uptake

T of radiolabeled bicarbonate by ICP in Autotrophic medium (50 mM FeSO4) suggests

that the type strain is able to fix CO2 from the energy derived from iron oxidation (P.L.

Siering, unpublished).

A. ferrooxidans ICPT appears to require a higher concentration of iron than EAO

isolates to grow autotrophically utilizing iron as an energy source. It is possible that the

EAO isolates may have a higher affinity for iron sequestering than strain ICPT. If strains

of EAO and ICPT were grown in mixed cultures, EAO isolates would prevail in lower

iron concentrations and ICPT strains would dominate in higher iron concentrations.

Alternatively, strain ICPT may have been isolated from an environment that was not as

oligotrophic as BSL. Previous studies often reference A. ferrooxidans as

‘heterotrophically-inclined’ due to the increased cell yields in the presence of organic

carbon or yeast extract (24). A clearer division between autotrophic and heterotrophic

lifestyles of strain ICPT may exist instead of being considered a true mixotroph. In order

T to obtain enough energy to fix CO2 and grow autotrophically, strain ICP may require

higher concentrations of iron. The minimal growth observed could have been a result of

carryover of organic molecules present in sub-cultures, initially inducing some heterotrophic growth.

On the other hand, EAO isolates (specifically EAO2 and EAO4) may truly be

Mixotrophs. Similar to the type strain, EAO1 rapidly oxidizes iron and reaches higher cell yields than the other EAO isolates in the presence of YE, but was unable to oxidize iron under autotrophic conditions (Fig. 17b). EAO2 and EAO4 actively oxidize iron both autotrophically and mixotrophically, with little difference in maximum cell yields obtained under these different growth conditions (Fig. 18c). There are a few possible explanations for these observations. First, isolates EAO2 and EAO4 may be oxidizing iron autotrophically to maintain basal metabolism similar to a VBNC state. Although cell densities do not increase, energy from iron may continue to drive active proton export to maintain intracellular positive electron potential for PMF and fix just enough CO2 to

maintain biosynthesis. Second, a small difference in maximum yields in iron-containing media (with or without yeast extract) may suggest that EAO isolates continue to fix CO2 even in the presence of an additional organic carbon source (e.g. yeast extract). Even though BSL receives allochthonous inputs of organic carbon, the system remains oligotrophic. The detection of RuBisCo I from members of Acidimicrobium suggests that

EAO isolates are capable of fixing CO2 in situ. The division between autotrophy and

heterotrophy is blurred because EAO isolates are adapted to continuous oligotrophy. A previous study of S. acidophilus (strain ALV) found that the mixotroph incorporated 20% of fixed-CO2 even when glucose was present (119). Yeast extract is a complex ingredient that contains organic carbon as well as growth factors and vitamins. Alternatively, the

slight enhanced growth in the presence of yeast extract may be due to the other

components present, not solely organic carbon. Since we did not observe an increase in

96 cell number when culturing in Autotrophic medium with iron, future studies will need to verify CO2 fixation by EAO isolates in autotrophic or mixotrophic conditions.

Ground rock pyrite was found to be a successful enrichment medium for isolation of Acidimicrobium (Table 3). Although the composition of the bedrock of BSL is not known, it is likely that pyrite is present and a source of iron in the thermal hot spring. At pH 2.0, soluble ferrous iron (~3.5 mM) and ferric iron (~1.5 mM) were present in 1% pyrite medium (Fig. 19). A. ferrooxidans ICPT type strain was unable to grow or oxidize iron present in pyrite (with or without yeast) (Fig. 16a, Table 8). Growth of the A. ferrooxidans ICPT strain on pyrite has been previously described as poor (24) but is improved in mixed cultures with other sulfur-oxidizing bacteria like Sulfobacillus (25).

Sulfobacillus strains are able to oxidize pyrite, thereby providing the higher concentrations of ferrous iron required for growth of strain ICPT.

Conversely, Acidimicrobium EAO isolates reached their highest cell densities (107 cells/ml) and growth rates in Pyrite medium, provided it was amended with 0.01%YE

(Fig. 18). The ferrous iron concentration present in the pyrite medium maybe closer to the optimal concentration for growth of EAO isolates rather than the slightly more inhibiting

10mM FeSO4 used in the growth and oxidation experiments (Fig. 17, 18). Alternatively,

EAO isolates might have been able to utilize sulfide within the pyrite as an energy source, or the pyrite particles themselves may act to adsorb inhibitory metabolites.

Given the necessity to generate ferric iron for the “ferric iron attack” on the mineral (see Chapter 1: Introduction - Iron and sulfur oxidation), it is puzzling that the presence of rapidly iron-oxidizing Acidimicrobium EAO isolates did not enhance the

dissolution of pyrite by increasing total soluble iron (Fig. 19). However, a decrease in pH

from 2.01 to ~1.78 was found in cultures of EAO after a 20 day period. The reduction in

pH was similar to cultures of Sulfobacillus isolates that did show an increase in total

soluble iron, suggesting that Acidimicrobium isolates are capable of pyrite oxidation.

Perhaps, the reason why an increase in soluble iron was not observed for Acidimicrobium

isolates but was evident in Sulfobacillus isolates may be due to their differing long-term survival strategies. After 2-3 days of growth, Sulfobacillus isolates form endospores and stop oxidizing iron (Fig. 17d), allowing ferrous iron to accumulate over a 20 day incubation period (Fig. 19b). However, Acidimicrobium does not form endospores and continues to oxidize iron even in the absence of growth (Fig. 17b, c). Therefore, ferrous iron does not increase over time as it is being oxidized to ferric iron, and the ferric iron binds to the pyrite mineral, resulting in no change of soluble of iron over the 20 day period. Long-term survival mechanisms for Acidimicrobium have not been previously investigated, and our results suggest that iron-oxidation in the absence of growth may provide enough energy for this acidophile to maintain homeostasis by actively pumping out protons in the acidic environment.

Cell morphology of Acidimicrobium

Cellular morphology and ultrastructures of Acidimicrobium have not been previously studied in detail. However, A. ferrooxidans ICPT was previously

characterized as small rod-shaped cells (0.35µm diameter) that can occur as filaments of

variable length under mixotrophic growth, while strain TH3 grew more commonly in

filaments under all growth conditions (24). Acidimicrobium EAO isolates also occurs as

small rods, and are generally straight and thin (0.3-0.4 µm). Interestingly, I noticed that

the A. ferrooxidans ICPT produced extremely long (10-25 µm) filaments only in the presence of iron (≥10 mM FeSO4) (Fig. 6), at stationary phase, and after accidental exposure to trace levels of nitric acid contained in residue from acid-washing of glassware. Many microorganisms produce filaments under stressful conditions such as oxygen deficiencies in aerobic granules in sequencing batch reactors (SBRs) (65) and heat stress in B. subtilis (121). Inhibition of growth and cell division (i.e. filamentous growth) of Bacillus subtilis is correlated with knockout of ftsH that encodes for energy- dependent metalloprotease as well as sensitivities to heat and salt stresses (121).

Motility of A. ferrooxidans ICPT was reported to occur during heterotrophic

growth on yeast extract (24). However, I noticed motility in the apparent absence of

flagella by both the type strain and EAO isolates during exponential growth in all

medium types. Interestingly, A. ferrooxidans ICPT contains genes encoding for flagella

and pili (26). For example, genes that encode for proteins such as FlgK a hook-associated

protein and CpaB a protein associated with pilus assembly. It is possible that flagella

might still exist in our Acidimicrobium isolates. Flagella are often too small to see in

phase contrast without the use of specialized stains. Preparation of samples for electron

microscopy was collected during early stationary phase in which motility was not

observed. Flagellar structures may have been disassembled during stationary phase or the

procedure to preserve the structure for visualization under electron microscopy may need

to be improved.

Ultra-structures of Acidimicrobium have not been previously reported, and this

study provides a first look as to the intracellular complexity of this genus. Due to the

composition of the envelope, TEM images of cells can be mistaken for Gram-negative

cells rather than Gram-positive (Fig. 8, 9). There appears to be a periplasm-like region

similar to a Gram negative cell, but this region contains a thick, layered cell wall instead

of a thin cell wall. As expected for a Gram-positive organism, the envelope appears to

lack an outer membrane. The envelope is similar to another Gram positive member of the

high G-C Actinobacteria – Acidothermus cellulolyticus (76). EAO isolates appear purple

when Gram stained (Fig. 5c), and SEM images of cell surfaces are smooth which is

characteristic of Gram positive bacteria (68). Protrusions from the cell wall surface (Fig.

7b, d) may be evidence of pili used for conjugation or nanowires used for electron

transfer. Yet, these protrusions were only found in a few images and may also be artifacts

from sampling and preparation.

Transmission electron microscopy for the Acidimicrobium EAO isolates and the

A. ferrooxidans ICPT revealed a single large inclusion within cells in both medium types

(Fig. 7, 8); this may function as an energy or carbon storage (68). Generally, inclusions

can vary in size, are often numerous within cells, and contain unique membranes (61).

The most common inclusions found in prokaryotes consist of poly-β-hydroxybutric acid

(PHB) that bond via ester linkages to form a long PHB polymer, eventually aggregating into granules (68). PHBs and other poly-β-hydroxyalkanoates (PHAs) are often synthesized in the presence of excess carbon suggesting a role in carbon storage. All

EAO isolates and the type strain were grown in the presence of carbon (in the form of

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yeast extract), leading to the possibility that these large inclusions are some type of PHA.

Also, cells collected for electron microscopy preparation were at the transition of

exponential to stationary phase, and this may explain why only a single granule or

absence of granules was observed in cells. It is also possible that the large inclusions are

processing.

The unknown particulates present in all EM images are indicative of mineral

oxides (possibly iron oxides) (77). All cultures contained iron that was autoclaved which

may have resulted in the formation of solid particulates (43). However further

investigation is needed to verify that these are not biological in origin. Alternatively,

particulates may be the result of other components found in the media. TEM grids were

post-stained with uranyl acetate and lead citrate. These positive stains appear as phase

dense and bind to phosphate and amino groups as well as other anions (91). Therefore,

phase dense particles do not necessarily indicate cellular components, but other

components that may be present in the media (i.e. organic wastes).

Caveats associated with growth measurements

Cell yields of 1x105 cells were considered minimal growth for all organisms and

conditions tested, and this was equivalent to cell yields obtained in negative controls

(with no added energy source present beyond what was contained in the inoculum) (Fig.

15 and 16). At this cell density and lower, sampling and microscopic counting errors

significantly increase as indicated by large vertical standard error bars (all growth figures); such errors make it more difficult to determine optimal conditions for growth. In

101 my growth experiments, I counted a maximum of 25 different fields per sample when cell counts were below 106/ml. One of the reasons why optical density methods were not used to determine cell yields in our study was due to the lack of visible turbidity in cultures and issues associated with measuring turbidity of cultures containing particulate iron oxides. Optical density (OD) methods require that cells absorb light in a concentration dependent manner and that a particular threshold of at least 106cells/ml is achieved to detect OD spectrophotometrically. Most of my studies did not meet this threshold. For our purposes, the direct count method was the best method available for cell counting.

Isolation and physiological characterization of Sulfobacillus

Strains of the genus Sulfobacillus are commonly found and isolated from geothermal environments, mineral sulfide mines, coal and mineral spoil heaps, and commercial metal leaching dumps (7, 17, 40, 120). Four species are currently classified in the genus Sulfobacillus: S. thermosulfidooxidans, S. acidophilus, S. sibiricus, and S. thermotolerans (14, 40, 75, 81). All strains of the genus Sulfobacillus have a mixotrophic

2+ 0 2- 2- metabolism on Fe , S , S4O6 , S2O3 , and sulfide minerals such as pyrite in the presence of low levels of yeast extract (0.01-0.2%). All strains are Gram positive and produce spherical endospores. Sixteen isolates obtained in this study shared 99% 16S rRNA nucleotide identity with S. acidophilus (Table 2).

There were similarities found among the physiologies of Sulfobacillus and

Acidimicrobium isolates. Optimal temperature and pH were similar between the two genera (Table 3). Autotrophic growth on iron, pyrite, elemental sulfur, or tetrathionate

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was not evident for either genus (Fig. 16, 17, 19, 20) as was previously shown for

representative of Sulfobacillus and Acidimicrobium (24, 81). However, both genera were

able to oxidize iron rapidly in the presence and absence of yeast extract (10mM

FeSO4/0.01%YE) (Fig. 17). There was no evidence for sulfur/tetrathionate oxidation in

the presence of yeast extract for either Acidimicrobium or Sulfobacillus, though we did

not run chemical analysis to ensure that no oxidation occurred. This was an unexpected

result for Sulfobacillus because sulfur oxidation is a hallmark phenotype for the genus.

Previous studies (81) indicated that a drop of culture pH from 3.0 to 1.67 was observed

whereas our studies did not observe a drop in culture pH. It is possible that the pH of the media we used for the sulfur oxidation studies was too low to observe a significant drop in pH (pH 2). Sulfur oxidation may have occurred but was undetectable by the methods used, and/or sulfur may be oxidized by another route. Further studies will be needed to verify this result as it would imply a new phenotype for Sulfobacillus.

Although there were physiological similarities found among Sulfobacillus and

Acidimicrobium, key differences may explain why Sulfobacillus can be cultivated more readily than Acidimicrobium. Doubling time for Sulfobacillus was often 2X faster than

Acidimicrobium in identical conditions (i.e. optimal temperature/pH/Heterotrophic medium) (Table 3). Organisms with a faster growth rate have an advantage when competing for similar limited resources (i.e. organic carbon). Sulfobacillus isolates can tolerate changes in temperature/nutrients because they have a wide temperature range for growth as well as the ability to produce endospores. Changes in environmental conditions, particularly temperature, may have occurred during sampling from BSL.

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There was a 24 hour delay before samples were incubated at optimum temperature in

which samples were left at ambient temperatures. The ability for Sulfobacillus to both

tolerate greater temperature fluctuations and enter a dormant state, may have provided an

additional advantage for Sulfobacillus strains to survive the process of sampling. In

addition, Sulfobacillus strains readily produce single colonies on solid media (57). A

single colony is often derived from a single cell and usually produces a characteristic

morphology type. Identification and purification of bacterial isolates is thus easily

obtained.

Using AIC analysis to differentiate between strains of Sulfobacillus and between

species of Sulfobacillus and Acidimicrobium

We attempted to utilize linear regression analysis to determine applicability of the analysis for use in physiological characterization of isolates. AIC analysis is commonly used in ecological studies to provide a non-subjective approach to estimate the effect

(magnitude) of a given variable on a response variable (73). For example, Bradford and colleagues (15) used AIC to investigate which independent variable (pH, temperature, etc.) affects respiration rates in soil bacteria. Use of multiple hypotheses testing for physiological studies has not been reported. Species identification using 16S rRNA is well established, but characterization studies still rely on subjective approaches to determine variation in strains. By including AIC analysis, I hoped to yield more robust estimates of strain variation. Due to the lack of full replication in samples, we were unable to make inferences on the interaction between pH and temperature for a given

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Sulfobacillus strain. However, we were able to determine that there was no difference

between interactions of pH or temperature on strains; inferring sub-species or ecotypes

could not be distinguished for pH and temperature alone. Given the low number of

replicates and similarity of growth rates across temperatures and pH (Table 3), I would

have arrived at the same conclusion. The models for distinguishing between species of

Sulfobacillus and Acidimicrobium were significant, thus accurately identifying species.

Results from AIC were not ground-breaking, but as an exercise for using multiple-model testing, it proved informative.

Isolation and physiological characterization of Alicyclobacillus

Alicyclobacillus strains are ubiquitous acidophiles found in soils and water of geothermal areas as well as nongeothermal soils (i.e. from gardens or woods), fruit juices, ores and wastewater sludge (21, 33, 35, 46, 62). The genus Alicyclobacillus contains 17 species in which the majority are easily distinguished from each other by phenotypic and chemotaxonomic characteristics (32). Alicyclobacillus species encompass a wide temperature and pH range for growth from 4-70˚C and pH 0.5-6.5, respectively, and all species produce ovoid endospores. Species of Alicyclobacillus can be categorized into three groups determined by growth temperature ranges: hyperthermophiles like Al. acidocaldarius ranging from 45-70˚C (opt. ~65˚C), thermophiles like Al. acidophilus ranging from 20-65˚C (opt. 40-55˚C), and mesophiles like Al. tolerans ranging from 4-

55˚C (opt. 35-42˚C). Most species of Alicyclobacillus appear to be strictly chemoorganotrophic. Two species (Al. tolerans and Al. disulfidooxidans) are

105 mixotrophic which resulted in the previously mistaken identity as unrelated Sulfobacillus species (S. thermotolerans and S. thermosulfidooxidans, respectively) (40, 59). During mixotrophic growth, ferrous iron, elemental sulfur and sulfide minerals can be utilized as energy sources in the presence of organic substrates or yeast. Most facultatively mixotrophic strains are sensitive to high concentrations of organic compounds and prefer mixotrophic growth conditions rather than growth on organic substrates alone (59). We obtained four isolates that share 99% 16S rRNA gene identity with Alicyclobacillus sp.

DSM6481 and A. tolerans (Table 2).

Optimum temperatures for growth of Alicyclobacillus isolates correlate with the average summer temperature of BSL and have a wide range of growth to include winter temperatures (Table 3) (103, 117). Although only one of the isolates I obtained in this study was unable to grow at the consistent pH of BSL (pH 2.0), other strains isolated in the lab have a wide pH range (not shown) (47). The doubling time and maximum biomass yield for Alicyclobacillus isolates was the greatest for all genera tested at their optimum growth temperature and pH (Table 3). However, Alicyclobacillus was monitored on a carbon-rich medium (1X PTYG) relative to the Heterotrophic medium that Sulfobacillus and Acidimicrobium were cultured in. Recently thawed frozen cultures of Alicyclobacillus isolates were unable to be sub-cultured onto FeSo plates and thus were maintained on 1X PTYG. For this reason, we did not analyze the ability of

Alicyclobacillus isolates to utilize different energy sources of iron and sulfur, even though the majority of isolates were related to mixotrophic A. tolerans. Some

Sulfobacillus isolates showed evidence of growth in 1X PTYG (not shown). It would be

106 interesting to re-examine and compare doubling times and maximum cell yields of

Sulfobacillus isolates in 1X PTYG medium. Unfortunately, Acidimicrobium isolates were unable to grow in 1X PTYG. As mentioned earlier, EAO isolates may prefer autotrophic and/or mixotrophic lifestyle for growth due to the oligotrophic conditions of BSL.

Overall, physiological parameters suggest that Alicyclobacillus strains are r-strategists that respond quickly to carbon inputs into BSL, and other students in the laboratory are investigating this hypothesis.

Incidentally, media with organic carbon will select for r-strategists from an oligotrophic habitat. Alicyclobacillus strains are inherently easier to cultivate since they have fairly rapid growth rates and produce single colonies on gelled-media containing

0.05% each of glucose and yeast extract (and organic N at 0.05%). However, only a few species of Alicyclobacillus are mixotrophs with the ability to utilize iron as an energy source (40, 59). Isolates we obtained in this study that were related to mixotroph A. tolerans produced white colonies (Table 2) and did not maintain viability on FeSo plates.

Speculation on Role of Acidophilic Isolates in BSL

Microbial communities in the environment are composed of interactive systems with multiple levels of organization (48). Physiological results from Acidimicrobium,

Sulfobacillus, and Alicyclobacillus isolates suggest that they may be active members of the community in BSL (Table 3), each with a unique role to play. The dominant presence of Acidimicrobium phylotypes in multiple SSU rRNA and RuBisCo gene clone libraries from BSL (11, 116, 117) suggests that Acidimicrobium are important members of the

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BSL community, and that may play important roles in primary production, iron and sulfur cycling. The ecological significance of the potentially rare members of the community, Sulfobacillus and Alicyclobacillus, should not be underestimated. These may not be well represented in previous clone libraries because of their ability to sporulate, thus evading lysis during DNA extraction procedures. Although the significance of rare members is often unknown, they may act as a “seed bank” of bacteria that become much more abundant when conditions in the environment changes (100). Here, I propose the roles and interactions of Acidimicrobium, Sulfobacillus, and Alicyclobacillus isolates in

BSL.

Acidimicrobium and Sulfobacillus are distantly related yet share similar physiologies. Isolates from both genera showed iron oxidation under autotrophic and mixotrophic conditions (Fig. 18). We did not monitor CO2 fixation by EAO and

Sulfobacillus isolates, however the detection of RuBiSCo I from Acidimicrobium in molecular surveys (102) indicate that it is possible. Sulfobacillus acidophilus has also been reported to contain genes in the Carbon Benson Cycle (i.e. cbbL) (19). Carbon fixation pathways for mixotrophic species of Alicyclobacillus are still unknown. The genome of Al. tolerans or Al. disulfidooxidans has not been sequenced and studies investigating presence of C-fixing genes have not been performed. Acidimicrobium may be able to utilize iron from BSL as an energy source for autotrophic/mixotrophic growth or for maintaining cell viability in the absence of growth. However, Acidimicrobium may not be able to provide enough organic molecules in the form of pyruvic acid to

108 heterotrophic members of the community. Instead, the significant role of Acidimicrobium may be continuous iron oxidation in the BSL.

Active iron oxidation by both genera may supply ferric iron to the system. At low pH (2.0), the majority of soluble iron present is in the form of ferrous iron. By providing

Fe3+ to the system, organisms that require ferric iron for anaerobic respiration (including

Acidimicrobium, Sulfobacillus, Al. tolerans, Acidicaldus organivorus) can be satisfied.

Also, ferric iron is considered the major oxidant for pyrite dissolution. Two Sulfobacillus isolates (JWO19m and JWO22m) and all Acidimicrobium isolates showed evidence for pyrite oxidization (Fig. 19, Table 8). The role of Sulfobacillus and Acidimicrobium in pyrite oxidation in situ may be significant since this activity results in the generation of reduced iron and intermediate redox state sulfur compounds which potentially can be used as energy sources by lithotrophic and mixotrophic members of the community.

Sulfobacillus strains have the potential to oxidize sulfur autotrophically and mixotrophically (81), however we did not find evidence for sulfur/tetrathionate oxidation in the Sulfobacillus isolates examined (Table 8). Considering that this is a hallmark phenotype for Sulfobacillus, further testing will be required to verify this result. Sulfur oxidation provides sulfate and sulfuric acid to the system. Sulfuric acid in BSL appears to be more dilute than other acidic habitats (34, 42, 117), suggesting that sulfur oxidation may not be as prevalent, or the generated sulfate is quickly reduced by sulfate respiring bacteria. Based on functional gene microarrays (102), there is a potential for acidophilic sulfate reducers to be present in BSL.

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Seasonal allochthonous carbon inputs carried by snow and rain runoff appear to be significant contributers to the organic matter present in BSL (117).

Chemoorganotrophs such as Alicyclobacillus isolates can grow rapidly and reach high densities in the presence of organic carbon (Fig. 11, 12 and Table 3). Based on functional gene microarrays and uptake of radiolabeled C sources, the BSL carbon cycle appears to be dominated by heterotrophic processes (102). Once the carbon is depleted in the system, Alicyclobacillus can sporulate and remain dormant until another massive input of carbon is received. Cells that do not sporulate might lyse and contribute an additional input of organic molecules to the system.

Many organic molecules produced by autotrophic organisms are self-inhibitory

(i.e. pyruvic acid and glycolic acid). Glycolic acid produced as a metabolite by autotrophic acidophiles has been recently shown to inhibit growth of Acidimicrobium ferrooxidans in mixed community reactors (79). The ability of Firmicutes (mainly

Sulfobacillus spp.) to metabolize glycolic acid may explain why Sulfobacillus spp. are the main iron-oxidizing bacteria in bioprocessing operations. Autotrophic bacteria may benefit from interacting with heterotrophic and/or mixotrophic acidophiles whom detoxify the environment by degrading the soluble organic wastes (e.g. glycolic acid) produced through autotrophic metabolisms.

Summary

We obtained three Acidimicrobium isolates (Table 2) representative of an abundant bacterial phylotype detected in multiple SSU rRNA gene clone libraries

110 prepared from BSL-derived DNA (11, 116, 117). Previous detection of RuBisCo I from

Acidimicrobium in BSL (102) suggests that this organism may also be important in primary production. The pH and temperature optima of Acidimicrobium EAO isolates correlate with the average summer temperature of BSL (~50˚C, pH 2) (Table 3), suggesting that these organisms are capable of active growth in BSL. Possible dormancy in the hotter >65˚C region of the lake (site D) and during the colder winter season needs further investigation. Acidimicrobium isolates EAO2 and EAO4 as well as Sulfobacillus isolates are able to oxidize iron autotrophically (Fig. 18), though only mixotrophic iron oxidation is correlated with cell growth; this suggests the energy yields from iron oxidation alone are insufficient to drive carbon fixation. However, energy generation through autotrophic iron oxidation may permit the maintenance energy needed for these acidophiles to survive in the absence of available DOC. It is possible that Acidimicrobium sp. EAO isolates may represent a new species of Acidimicrobium or a new ecotype based on the comparative characterization between EAO isolates and the type strain. However, further work would be required to produce evidence to support a proposal for a new species. It is likely that Acidimicrobium are important players in the carbon and iron cycles of BSL.

This study also provided the first detailed ultrastructure descriptions for the

Acidimicrobium genus. The cellular envelope appears more complex than many other

Gram-positive bacteria. The cell wall is 30-60 nm thick and composed of multiple layers

(Fig. 7). In addition, presence of putative PHAs was observed in the presence of even low

111 concentrations (0.01%) of yeast extract. Further investigations of unidentified nanoparticles will be required to verify that they are not biotic in origin.

In addition to Acidimicrobium, we isolated 16 strains closely related to

Sulfobacillus acidophilus and four isolates closely related to Alicyclobacillus sp. and A. tolerans (Table 2). Temperature and pH ranges and optima of Sulfobacillus and

Alicyclobacillus isolates suggest that these members of the community are also active in

BSL with significant putative roles in the ecosystem. Sulfobacillus isolates are able to oxidize iron and some are able to oxidize pyrite in the presence of yeast extract.

During inputs of allochthonous material from snow melts or rain runoff, it is possible that Alicyclobacillus and other heterotrophs rapidly use the carbon and return the system to an oligotrophic state. High concentrations of organic carbon can be inhibitory to autotrophic acidophiles (79), and thus Alicyclobacillus or Sufobacillus may help detoxify the environment for Acidimicrobium and others.

Overall, the significance of cultivating a potentially dominant primary producer from BSL is paramount to understanding ecosystem processes. Rarely is the dominant member of a community isolated since cultivation favors the rare and opportunistic members (100). With access to Acidimicrobium in culture, and knowledge of its optimal growth conditions, we can begin to verify its functions in the BSL community.

Future Work

Future studies are needed to verify autotrophic or mixotrophic growth for both

Acidimicrobium and Sulfobacillus isolates on iron substrates. Evidence of iron oxidation

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coupled to growth in the presence (but not absence) of yeast, suggests mixotrophic (rather

than autotrophic) growth. However isolates may not be utilizing the organic carbon from yeast extract, rather other components present like growth factors or vitamins. Further

14 studies of CO2 fixation via radioisotope uptake assays of C-bicarbonate would verify

autotrophy of isolates. Also, the utilization of real-time PCR for quantifying gene

expression of Acidimicrobium RuBisCo I (i.e. cbbL) can also be used to verify autotrophy. Future studies are also needed to quantify sulfur loss and the gain of oxidized forms of sulfur during growth to conclude whether the isolates obtained in this work are capable of sulfur oxidation.

SSU rRNA gene clone libraries suggest that Acidimicrobium is an abundant member of the BSL community. Further studies utilizing real-time qPCR or FISH can also be used to quantify Acidimicrobium in BSL to verify its abundance. Previous studies using FISH from environmental samples from BSL were difficult due to the background fluorescence from metals. A real-time qPCR approach may prove to be more efficient and has been utilized with great success in quantifying species-specific densities from environmental water samples (23).

Continued isolation and characterization of Acidimicrobium-like organisms will increase our understanding of the functional taxonomic groups involved in the BSL ecosystem. Isolation methods should include enrichment of samples in 1% Pyrite,

Autotrophic medium adjusted to 10mM FeSO4, and Mixotrophic medium at 45 and 50˚C.

Subsequent isolation and purity might be established by serial dilutions in ½ increments

as well as sub-culturing onto FeSo and FeSo overlay at various iron concentrations

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(25mM, 10mM, and 1mM), under aerobic and microaerobic conditions. Orange colonies indicative of iron oxidation as well as orange smears should be screened with

Acidimicrobium-specific primers used in this study. All culturing work should correlate with careful observation under 400 and 1000X phase contrast microscopy for thin rods of variable length (lacking endospores). Multiple fields of view per sample will be necessary to identify these slow growing bacteria at low cell densities (4 cells/field; 106 cells/ml).

By investigating the mechanisms for long-term survival of EAO, especially under conditions that are not conducive for growth, we may better understand the seasonal role of Acidimicrobium in BSL. Commonly used methods for detection of cells in the VBNC state include direct microscopic examination for the presence of an intact cytoplasmic membrane (using the BacLight® Live/Dead assay) or reverse transcriptase (RT)-PCR to detect genes expressed by VBNC cells (Oliver 2009). In addition, in order to verify that

EAO isolates are not capable of growth during BSL winter temperatures, we will need to monitor growth between the last observed minimum temperature of growth of 45°C and the next temperature in succession were no growth was observed at 35°C (eg. 37, 39, 41,

43°C). It would also be interesting to examine iron oxidation by EAO isolates at these lower temperatures to determine if they may still be able to maintain viability from the energy acquired from iron oxidation.

The characterization studies performed in this work were focused on identifying parameters that enable optimal growth. Further characterization studies can include metal and metabolite toxicities that would focus on factors that inhibit growth. For example, a

114 metabolite toxicity study would begin by determining the composition of organic exudates from autotrophic isolates (e.g. glycolic acid or pyruvic acid) which have been shown to be self-inhibiting (79). In order to investigate sensitivities to organic exudates, the isolates can be screened against glycolic acid by inoculating organisms in increasing concentration of the exudate. Organisms that were positive for growth would be tested for the ability to metabolize glycolic acid by comparing growth of isolates inoculated into media amended with glycolic acid or without.

115

APPENDIX

Appendix A.

Freezing cultures. Freezer stocks of isolated strains were established for

Sulfobacillus and Alicyclobacillus isolates by streaking cultures for confluent growth onto

FeSo plates, and incubating at 50˚C for 2-5 days. Once growth was observed, cultures

were aseptically transferred by a metal loop to liquid medium 0.1X PTYG (pH 5.0)

containing 20% (v/v) glycerol. Three 533 μL aliquots of each strain were prepared in

2ml screw cap tubes and stored in the -80°C freezer. After one month of freezing, strains

were checked for viability by emptying the entire contents of a single frozen tube of each

strain onto FeSo or 1X PTYG (pH 2.9) plates and incubating for up to 5 days at 50˚C.

Acidmicrobium isolates were obtained from plates and each grown in two 50 ml serum vials containing liquid heterotrophic medium (15-20ml each) at 45˚C until cell densities reached at least 20 cells per field at 400X (phase contrast microscope). Cultures were centrifuged at 9000X g (RC5C Sorvall Instruments DuPont fixed angle rotor SA-

600) for 10 minutes. Centrifuged cultures were immediately transferred to an ice bucket, and supernatant was removed aseptically; pellets were resuspended in ~1.6ml heterotrophic liquid medium (pH 5.0) containing 15% (v/v) glycerol. Both 1.6ml suspensions of the same isolate were pooled, and 250µl increments were distributed to sterile cryogenic vials. Vials were stored at -80˚C. Validation of viability of frozen cultures occurred at 2 wks, followed by 1 year intervals after freezing by adding 100ul to

116

liquid heterotrophic medium and the rest of the tube contents (~150µl) onto FeSo plates,

incubated at 50˚C.

Appendix B.

Maintenance of cultures. Sulfobacillus and Alicyclobacillus isolates are sub-

cultured every 3-4 weeks onto either FeSo or 1X PTYG pH 2.9 plates, respectively, and

stored in the fridge. Plates are incubated at 50˚C for 2-5 days in sealed plastic containers.

Isolates JWO16m-JWO22m grow poorly aerobically, therefore they continue to be

incubated microaerophilically (BD* Diagnostic anaerobic jars and Mitsubishi

AnaeroPak*-Microaero, Mitsubishi Gas Chemical, Company, Inc.). Sulfobacillus isolates are also capable of growing in 1X PTYG liquid and plates (pH 2.0-2.9). Colony morphologies on 1X PTYG appear as white flat circles. Even though most

Alicyclobacillus strains were isolated from FeSo plates, recently thawed frozen stocks were not capable of growing on FeSo plates.

Acidimicrobium isolates are maintained in Heterotrophic and Mixotrophic liquid media as well as FeSo plates adjusted to 10mM FeSO4. Liquid cultures of 10 ml volumes

in sealed Hungate tubes are incubated at a slanted position at 50˚C and the headspace is

replenished with 0.2 µm filter-sterilized air every 1-2 weeks. Every 3-4 weeks, isolates are sub-cultured from freshly grown (~3-5 days) FeSo plates and sub-cultured into fresh liquid media. Plates of FeSo are sub-cultured every 3-4 weeks from Mixotrophic liquid medium (100µl). After 2-3 days, inoculated Mixotrophic medium turns yellow indicating active iron oxidation. Plates are incubated at 50˚C microaerophilically for 5 days, and

117

stored in the fridge. Acidimicrobium isolates are capable of growing aerobically, but

consistently grow better under microaerobic conditions. Growth on FeSo plates adjusted to 1mM produce white flat colonies. However, EAO isolates were unable to grow in 1X

PTYG liquid or plates (pH 2.0-2.9).

Appendix C. Statistical equations used for linear regression modeling (5). To rank models, we used Akaike Information Criterion (AICc) for small sample sizes as

Equation 2:

2 ( + 1) = 2 (L( )| ) + 2 + 1 𝐾𝐾 𝐾𝐾 𝐴𝐴𝐴𝐴𝐴𝐴𝐶𝐶 − 𝑙𝑙𝑙𝑙𝑙𝑙 𝜃𝜃 𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 𝐾𝐾 𝑛𝑛 − 𝐾𝐾 − where K is the number of parameters included in the model. The log-likelihood of the model given the data, log(L(θ)|data), reflects the overall fit of the model. Smaller values indicate worse fit. AIC penalizes for the addition of parameters, thereby selecting a model that fits well but has a minimum number of parameters. To correct for the bias from too many estimated parameters in relation to the size of the sample, this second order variant of AIC includes the effective sample size n. If n is large (asymptotic) with respect to K, the second-order correction is neglible and AICc converges to AIC.

Therefore, the use of AICc is highly recommended in practice over AIC.

Equation 3:

= 2 (L( )| ) + 2

𝐶𝐶 Delta AIC (∆i) and Akaike𝐴𝐴𝐴𝐴𝐴𝐴 weights− 𝑙𝑙𝑙𝑙𝑙𝑙 (wi) can𝜃𝜃 be𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 used to compare𝐾𝐾 models. The delta

AIC is a measure of each model relative to the best model and is calculated as

118

Equation 4: ∆i = AICi – min AIC

Where AICi is the AIC value for model i, and min AIC is the AIC value for the best

model. A change < 2 suggests substantial evidence for that model; 3-7 suggests the

model has considerably less support; and > 10 indicates the model is very unlikely.

Akaike weights represents the ratio of the delta AIC of a given model relative to

the whole set of R candidate models, and is calculated as (p.88)

Equation 5:

1 exp i = 2 1 =1 exp�− ∆2� r 𝑅𝑅 ∑𝑟𝑟 �− ∆ �

Where ∆r represents delta AIC for the whole set of r models and ∆i represents delta AIC

of model i. This normalizes delta AIC to compare them on scale of zero to 1 (i.e. the sum

of the wi equals 1). Akaike weights indicate the probability that the model is the best among the whole set of candidate models.

119

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