THE INFLUENCE OF O2 AVAIABILITY ON THE GROWTH OF FE(III) REDUCING BACTERIA IN COAL MINE-DERIVED ACID MINE DRAINAGE
A Thesis
Presented to The Graduate Faculty of the University of Akron
In Partial Fulfillment of the Requirements for the Degree Master of Science
Zachary Santangelo August, 2019 THE INFLUENCE OF O2 AVAIABILITY ON THE GROWTH OF FE(III) REDUCING BACTERIA IN COAL MINE-DERIVED ACID MINE DRAINAGE
Zachary Santangelo
Thesis
Approved: Accepted:
______Advisor Interim Dean of the College Dr. John Senko Dr. Linda M. Subich
______Faculty Reader Dean of the Graduate School Dr. Teresa Cutright Dr. Chand Midha
______Faculty Reader Date Dr. Linda R. Barrett
______Department Chair Dr. David N. Steer
II ACKNOWLEDGEMENTS First, I would like to thank my advisor, Dr. John Senko, for pushing me and making me think about everything from every point of view. His research and ideas helped me think critically about research I never even considered. Dr. Senko constantly helped me become the best writer I could possibly be. I will always appreciate that in my work in the future.
The members of my committee: Dr. Teresa Cutright and Dr. Linda Barrett, I offer my sincerest gratitude. I had a couple classes with Dr. Cutright, both of which I repeatedly thought back to when approaching my own research. She helped me not just think about problems, but truly understand what was happening. Dr. Barrett, I sincerely thank for her time in helping me move forward with my thesis, and making sure I knew what I was trying to explain properly.
I must offer my thanks to Tom Quick. Without Tom, I surely would still be in the lab trying to get the dionex to work. Additionally, I would like to thank Elaine Butcher, who helped me with countless office questions as well as just enjoying conversation.
Tom and Elaine deserve more thanks than I could possibly give them.
Andrea Fodorne Mathe, from the Ohio State University, thank you for your help with the SEM. Setting up time and analyzing my samples with me not once but twice,
Andrea was very helpful in helping me work with the machine that allowed me to retrieve images of the bacteria in my samples.
To my fellow students, I thank you for all the time we spent working and laughing together. Bobby Miller and Shagun Sharma, thank you for your constant help and support
III in the lab. With their help, I was able to learn how to use most equipment in the lab. With their support, I was able to confidently work on my research. Kayla Calapa, thank you for your help and answering my many questions. Nick Wander, R.J. McGinnis and Rebeccah
DiPuccio, thank you all for the time you spent with me. These three had endless conversations with me that helped me keep my head on straight, and think my way out of any problem. Tim Schmucker, thanks for helping me back to reality time and again. I owe so much to every one of you.
To Valerie Miller, thank you for your constant support. Valerie followed me to
Akron without hesitation. Without her, none of this would have been possible. She knows how to get me fired up and take challenges head on, while also cooling my jets when I overreact. Our life continues to change and I never regret it. We continue moving forward, and while the future is uncertain, I remain excited to see it through with you.
Finally, to my friends and family, I thank for their support. No matter where they were, I was constantly shown the love and support I needed to push through everything.
Mom and Dad, you never doubted me, and continue to push me to do what I dream. I will continue to dream, thanks to them. Nick, Andrew, Luke, and Natalia, I wish I could have spent more time with all of you these past two years. So much has happened and you have all grown in ways I truly wish I could have been there for. My siblings have always been there for me, and I will always be there for them. Owen, Brendan, and Cece, thanks for your support and literally being here for me. I could not ask for better friends.
Everyone has shown me nothing but love, support, and pride these past two years. It is thanks to all of you, I have been able to accomplish my goals.
IV ABSTRACT
Acid mine drainage (AMD) is an environmentally harmful outcome of coal mining. Mining exposes FeS2 to oxygen and results in low pH and iron oxidation. Fe(III) precipitates after Fe(II) is oxidized in AMD fluids that have become exposed to a combination of oxygen (O2) and water (H2O). Bacteria in AMD acquire energy through respiration. Aerobic respiration uses the most favorable terminal electron acceptor, oxygen. Once oxygen is depleted, the next thermodynamically favorable terminal electron acceptors can be used, including Fe(III) and sulfate. Fe(III) reducing bacteria are anaerobes, respiring in anoxic conditions. However, recent studies indicated that Fe(III) reduction occurred in oxic conditions. I hypothesized that Fe(III) reducing bacteria would not be inhibited by oxygen, under acidic conditions. Enrichment cultures in anoxic and oxic conditions with Fe(III)-containing media were tested for 159 days. Cultures were inoculated with material from an AMD contaminated site. Cultures were maintained through three transfers. After each transfer, Fe(III) reduction was observed by quantifying the accumulation of Fe(II). Fe(III) reduction occurred in both oxic and anoxic conditions. Culture samples were examined under a scanning electron microscope
(SEM), where rod-shaped bacteria were observed in both anoxic and oxic cultures. 16S rRNA abundance of sequences indicates that Bacilli were the majority of the Fe(III) reducing bacteria in oxic cultures and Clostridia were the majority of the Fe(III) reducing bacteria in anoxic cultures. In acidic conditions, Bacilli reduced Fe(III) without being inhibited by oxygen.
V TABLE OF CONTENTS List of Figures...... vii List of Tables………………………………………………………………….………..viii Chapter I. Introduction……………………………………………………………………………..1 1.1 Acid Mine Drainage…………………………………………………………..1 1.2 Oxidation-Reduction Reactions and Terminal Electron Acceptors…………..2 1.3 Iron Mounds…………………………………………………………………...5 1.4 Previous Studies……………………………………………………………….7 1.5 Hypothesis……………………………………………………………………..8 II. Materials and Methods...……………………………………………………………...10 III. Results..……………………………………………………………………………....16 3.1 Approach……………………………………………………………………..16 3.2 First Round…………………………………………………………………..19 3.3 Second Round………………………………………………………………..21 3.4 Third Round………………………………………………………………….22 3.5 Scanning Electron Microscopy………………………………………………26 3.6 Taxonomic Composition of Cultures………………………………………...34 IV. Discussion..…………………………………………………………………………..37 4.1 Fe(III) Reduction in AMD…………………………………………………...37 4.2 AMD Sulfate Reduction……………………………………………………..41 4.3 Change in pH………………………………………………………………...42 4.4 Bacteria Abundances………………………………………………………...44 4.5 Iron Mound Applications…………………………………………………….45 V. Conclusion...………………………………………………………………………….47 References….…………………………………………………………………………50
VI List of Figures
1 Photograph of AMD flowing over the iron mound at the Mushroom Farm..…6
2 Culture transfer process……………………………………………………...12
3 Fe(II) concentrations, sulfate concentrations, glucose concentrations, and pH
values of the anoxic and oxic cultures over 159 day period. ………………..17
4 SEM of the uninoculated medium that contains Fe(III) hydroxide particles...27
5 SEM of an oxic culture……………………………………………………...28
6 SEM of an oxic culture………………………………………………………29
7 SEM of an anoxic culture……………………………………………………30
8 SEM of a culture in a medium consisting of glucose, but no Fe(III)………...31
9 16S rRNA gene abundance of sequences at the class-level………………….36
VII List of Tables 1 Table of Redox couples……………………………………………….…………..3
VIII CHAPTER I
INTRODUCTION
1.1 Acid Mine Drainage
Acid mine drainage (AMD) is the product of sulfide-bearing material that is
exposed to a combination of oxygen (O2) and water (Akcil and Koldas, 2004). Oxygen
oxidizes sulfide minerals (Eq. (1)).
2- 2+ + FeS2 + 3.5 O2 + H2O → 2 SO4 + Fe + H (1)
Pyrite is a mineral commonly associated with coal seams (Akcil and Koldas, 2004). This
process can occur naturally, but mining enhances the sulfide minerals exposure to oxygen
(Akcil and Koldas, 2004). The most common metal sulfide exposed in mining is pyrite
2+ 2- + (FeS2) (Baker and Banfield, 2003). Sulfur oxidation (Eq. (1)) yields Fe , SO4 , and H ,
contributing to the amount of total dissolved solids already within natural waters (Akcil
and Koldas, 2004). AMD has high concentrations of sulfate and iron from oxidation of
sulfide-containing minerals of mines (Kusel et al., 1999). The characteristics of AMD
include low pH, high conductivity, and high metal concentrations (Eby, 2004).
2+ + 3+ 14 Fe + 3.5 O2 + 14 H → 14 Fe + 7 H2O (2)
Fe(II) oxidation by oxygen (Eq. (2)) reacts at a relatively slow rate at low pH (Baker and
Banfield, 2003). Bacteria can accelerate Fe(II) oxidation (Baker and Banfield, 2003).
Fe(II) oxidation will increase the concentration of Fe(III) produced (Eq. (2)) (Akcil and
Koldas, 2004). Fe(III) may become reduced, to oxidize additional pyrite-S (Eq. (3))
(Akcil and Koldas, 2004).
3+ 2+ 2- + FeS2 + 14Fe + 8H2O → 15Fe + 2SO4 + 16H (3)
1 Sulfide and Fe(II) will serve as electron donors when used for respiration by microorganisms (Sawyer et al., 2003). Fe(II) oxidizing bacteria use oxygen as a terminal electron acceptor (Eq. (2)) in order to create the energy needed for cell synthesis and maintenance (Sawyer et al., 2003). However, AMD that has high pH (5.5-8.0) will be unsuited for bacteria that prefer acidic conditions. AMD enters circumneutral streams and high pH enhances the rate of abiotic Fe(II) oxidation and subsequent hydrolysis of Fe(III) and precipitation of Fe(III) hydroxides (Eq. (4)), hydroxides that are known as
“yellowboy” (Akcil and Koldas, 2004; Senko et al., 2008).
3+ + Fe + 3H2O → Fe(OH)3 + 3H (4)
The precipitation of Fe(III) hydroxides and production of hydrogen ions (Eq. (4)) will cause circumneutral streams to become highly acidic. As more Fe(III) hydroxides are produced, an accumulation of Fe(III) hydroxides in AMD impacted streams (Brantner et al., 2014). When both Fe(II) and oxygen are available, Fe(II) oxidation will continue to occur and produce Fe(III) hydroxides (Burwick et al., 2017). A trait that is advantageous to remediation is Fe(III) hydroxides can be physically removed from AMD as a sludge
(Akcil and Koldas, 2006). The increasing abundance of Fe(III) hydroxide phases will result in large Fe(III) crust deposits, termed “iron mounds” (Senko et al., 2008; Gouin et al., 2013; Burwick et al., 2017).
1.2 Oxidation-Reduction Reactions and Terminal Electron Acceptors
A spontaneous process will proceed in the direction that produces the highest energy for work available (Sawyer et al., 2003). Certain conditions provide different
2 energy changes that are related to the equilibrium of all chemical reactions (Sawyer et al.,
2003). The standard reduction potential is a measure of the tendency of chemical species to gain or lose electrons (Willey et al., 2009). The use of a terminal electron acceptor is dependent on the amount of energy that can be obtained. When comparing the different terminal electron acceptors, oxygen is the most thermodynamically favorable terminal electron acceptor (Table 1).
Table 1. A higher potential (volts) number indicates a more favorable terminal electron acceptor. Potentials were calculated with standard values found in Eby, 2004.
Redox Couples Potential (Volts)
CO2/CH4 0.15
2- - SO4 /HS 0.25
2+ Fe(OH)3/Fe 1.06
2+ MnO2/Mn 1.23
O2/H2O 1.23
Respiration is the energy-yielding process that uses the transfer of electrons to produce adenosine triphosphate (ATP) (Willey et al., 2009). Respiratory chemoorganotrophs generate ATP, energy for the cell, by oxidation-reduction reactions involving organic carbon as electron donors and a separate terminal electron acceptor
(Willey et al., 2009). Lithotrophs use inorganic compounds as an electron source (Willey et al., 2009). Respiration can be done aerobically or anaerobically. In aerobic respiration,
3 oxygen is the terminal electron acceptor. In anaerobic respiration, a terminal electron acceptor other than oxygen is used for the terminal electron acceptor (Willey et al., 2009).
When electrons are transferred from the donor to the terminal electron acceptor, energy, as ATP, is captured from exothermic reactions (Eby, 2004) ATP is synthesized, then breaks down to ADP, releasing the energy needed for endothermic reactions to occur
(Willey et al., 2009).
Biological oxidation-reduction reactions will first occur where there is the greatest difference in redox potential between electron couples, such as using organic carbon as an electron donor with oxygen as an electron donor (Eq. (5)) (Eby, 2004).
C6H12O6 + 6O2 → 6CO2 + 6H2O (5)
- Other electron acceptors besides oxygen include NO3 , sulfate, and Fe(III) (Willey et al.,
2009).
2- 2- 3SO4 + C6H12O6 → 3S + 6H2O + 6CO2 (6)
Dissimilatory sulfate reduction is a type of anaerobic respiration using sulfate as the terminal electron acceptor (Eq (6)) (Willey et al., 2009). Sulfate reduction is not as thermodynamically favorable as or Fe(III) reduction (Table 1).
Fe(III) reduction (Eq. (7)) is one of the most significant reactions that takes place in anaerobic sediment (Lovley et al., 1993).
- 2+ C6H12O6 + 24 Fe(OH)3 + 42 H+ → 6 HCO3 + 24 Fe + 60 H2O (7)
Fe(III) reduction oxidizes organic matter in anoxic conditions (Lovley et al., 1993).
Fe(III) reduction will occur in AMD streams under microaerophilic and anoxic conditions (Baker and Banfield, 2003). While uncommon, some anaerobes are capable of
4 2- metal reduction when exposed to oxygen (Lin et al., 2003). When SO4 and Fe(III) are
being reduced in the presence of oxygen, bacteria are not using the most
thermodynamically favorable terminal electron acceptor. It is not entirely understood
why or how often this occurs.
1.3 Iron Mounds
Fe(II) oxidation occurs as AMD flow emerges at the terrestrial surface and
flows over soil, precipitating and removing Fe(III) from the stream. Fe(III) hydroxide
deposits over time, from AMD build up, creating iron mounds (Gouin et al., 2013). Iron
mounds are large deposits, tens to hundreds of cm deep, of Fe(III) hydroxides (Brantner
et al., 2014). An iron mound has been measured to be accumulating as fast as 0.4-0.5
cm/year at a location in eastern Ohio known as “The Mushroom Farm” (Brantner et al.,
2014) (Figure 1).
5 Figure 1. Photograph of AMD flowing over the iron mound at the Mushroom Farm, (Courtesy of Dr. John Senko).
6 The water flowing over the iron mounds is 0.5-1 cm deep (Gouin et al., 2013). As the flow of AMD continues, the Fe(II) concentration and pH decrease (Gouin et al., 2013).
Lower Fe(II) concentration is another indication of Fe(II) being oxidized, and Fe(III) hydrolysis producing iron mound. The production of iron mounds is mainly a biological process caused by Fe(II) oxidizing bacteria (Brantner et al., 2004). As Fe(II) oxidizing bacteria produce Fe(III) (Eq. (2)), oxygen concentrations that support Fe(II) oxidation are depleted. These microorganisms bury themselves in rising Fe(III) (Brantner et al., 2014).
The oxygen depleted zones within iron mounds leads to anaerobic activities. Fe(II) oxidation is favorable in aerobic conditions, but Fe(III) reducing bacteria metabolize in anaerobic conditions, such as within the depths of iron mounds.
Previous Studies
In previous research, Fe(II) oxidizing bacteria and Fe(III) reducing bacteria were quantified alongside oxygen concentrations (Brantner et al., 2014). For Brantner et al.
(2014), dissolved oxygen (DO) concentrations were consistent, about 80 µM, in the top 2 cm of sediment; below 2 cm the DO concentration decreased with increasing depth. Iron mound systems with similar oxygen distributions will have aerobic conditions at the surface of the soil and anoxic conditions below the surface. Fe(II) concentration increased below the sediment-water interface, indicating Fe(III) reducing bacteria activity increased with depth (Brantner et al., 2014). However, maximum levels of Fe(III) reducing bacteria were measured within the top 2 cm of the cores, in oxic conditions.
Brantner et al., (2014) produced evidence of Fe(III) reducing bacterial activities in oxic conditions.
7 In laboratory incubations, a stable Alicyclobacillacea culture, A06, was incubated with Fe(III) (Burwick et al., 2017). As depth in the cultures increased, DO decreased from 300 to 1 µM, like Brantner et al. (2017), indicating anoxic conditions increase with depth in AMD (Burwick et al., 2017). Fe(III) reduction was observed in shallow, oxic conditions within the experimental cores (Burwick et al., 2017). A06 was capable of reducing schwertmannite-Fe(III) in the presence of oxygen (Burwick et al., 2017). Fe(III) reduction was still most prevalent in the anoxic locations deeper in the cores (Burwick et al., 2017). Burwick et al. (2017) concluded that Fe(III) reducing bacteria were capable of reducing Fe(III) in oxic conditions, but the production of Fe(II) was lessened by the presence of oxygen.
Hypothesis
The experiments by Brantner et al. (2014) and Burwick et al. (2017) both indicated oxygen did not completely stop Fe(III) reduction from occurring. I experimented with two systems, one anoxic and one oxic. The microorganisms came from Fe(III) hydroxide soil previously collected at Mushroom Farm, in North Lima,
Ohio. Using the iron mound sediment as inoculum for enrichments, I focused my research on Fe(III) reduction in order to observe how successfully biotic Fe(III) reduction occurs in oxic conditions.
Hypothesis: Fe(III) reducing bacteria would not be inhibited by oxygen, under acidic conditions. If Fe(III) reduction continues in both settings, then AMD remediation work could be undone by Fe(III) reducing bacteria. Fe(III) reduction is an anaerobic process (Eq. (7)). Based on thermodynamic favorability of terminal electron acceptors
8 (Table 1), Fe(III) should not be reduced until oxygen is depleted. However, the difference in redox potential could be close enough that Fe(III) reducing bacteria become energetically competitive under acidic conditions (Kusel et al., 1999). Fe(III) reduction will yield 0.77 V while oxygen reduction will yield 0.82 V (Kusel et al., 1999). I determined if there is a difference in the rate of Fe(III) reduction enriched from iron mound sediments under anoxic and oxic conditions. If Fe(III) reducing bacteria successfully reduce Fe(III) in both anoxic and oxic conditions, then there will be evidence of Fe(III) reduction independent of oxygen availability.
9 CHAPTER II
MATERIALS AND METHODS
To determine if oxygen inhibits Fe(III) reduction, enrichment cultures were created to study Fe(III) reducing bacterial activity in both anoxic and oxic conditions.
Anoxic conditions do not contain oxygen. Oxic conditions contain oxygen. Initial oxic and anoxic cultures were inoculated with sediment from Mushroom Farm, collected in
2016 and stored at 4oC.
The medium was composed of C6H12O6 (5 mM), Fe(SO4)3 (25 mM), (NH4)2SO4
(10 mM), MgSO4 (2 mM), tryptic soy broth (TSB; 0.25 g/L), vitamins and trace metals
(Burwick et al., 2017). Fe(III) from the Fe(SO4)3 in the medium was the source of
Fe(OH)3 precipitation (Eq. (4)). The pH was adjusted to 4.5 using 1 M H2SO4 and NaOH as needed. The volume was brought up to 1 L and mixed thoroughly. The medium was bubbled with N2 for 45 minutes to remove oxygen. In an anaerobic chamber, 50 mL of the medium was then dispensed into media bottles, which were sealed with thick rubber stoppers and held in place with aluminum crimp seals. The media bottles were sterilized by autoclaving. Iron mound samples have been previously collected from the Mushroom
Farm and were used as the inoculum for the enrichments. 0.5 g of Mushroom Farm sediment was added to every 1 mL of sterilized SAMD. SAMD was composed of CaSO4
(5 mM), MgSO4 (4 mM), and Na2SO4 (1 mM) (Burwick et al., 2017). The solution of sediment and SAMD was used for the initial inoculation. 5 mL of inoculum, 10% of a solution, was used for each incubation. All cultures were incubated and remained at room temperature. The oxic cultures were injected with 180 mL of atmospheric air, containing
10 about 20% oxygen. The anoxic and oxic enrichments were conducted in triplicate. There were uninoculated controls. Once the enrichment culture reached stationary phase (as indicated by no further Fe(II) accumulation), bacteria were transferred to fresh media
(Figure 2).
11 Figure 2. Culture transfer process. The initial culture was created using soil from Mushroom Farm, indicated by orange. Once a culture reaches the stationary growth phase, it was transferred into a media bottle with fresh media.
12 All sampling was collected in the anaerobic chamber. To identify if Fe(III) reduction was inhibited by the presence of oxygen, Fe(II) concentrations were measured.
If Fe(II) production was less in oxic conditions than in anoxic conditions, considering standard deviation, than it would be evident Fe(III) reduction was inhibited by oxygen.
Fe(II) and sulfate concentrations were quantified every 3 days. Fe(II) was measured with the ferrozine assay (Stookey, 1970). Samples were measured for soluble Fe(II), diluted
1/5 with HCl. Next, 0.20 mL of a sample was taken and injected into a microcentrifuge tube. The sample was centrifuged on maximum speed for 5 minutes. 0.10 mL of the supernatant was injected with 0.40 mL of 0.5 M HCl (Lovley and Phillips,1987).
Samples were taken for total Fe(II). 0.10 mL of sample from the culture was injected into an empty microcentrifuge tube. 0.40 mL of 0.5 M HCl was added into the microcentrifuge tube. The microcentrifuge tube was centrifuged at the maximum speed for 5 minutes. 0.40 mL of the supernatant was then transferred to a new microcentrifuge tube. A ferrozine assay was used for measuring Fe(II) (Stookey, 1970). The reagents used were 1 g ferrozine reagent in 1 L of 50 mM HEPES buffer (pH adjusted to 6.8), 1 M HCl, and Fe(II) standards: 0 mM, 0.025 mM, 0.1 mM, 0.25 mM, 0.5 mM, and 1 mM.
Ferrozine solution was stored out of light. 0.02 mL of standard were dispensed into a disposable cuvette. 1 mL of ferrozine solution was added to the cuvette. Absorbance was read at 562 nm in the spectrophotometer. A standard curve was made in excel using the absorbance (A562) on the x axis and Fe(II) concentration on the Y axis (y=mx). m was the slope. X was A562. Y was Fe(II) concentration. Fe(II) concentration was calculated by multiplying A562 (x) by the slope (m). The same process is used for the unknown Fe(II)
13 concentrations, replacing the 0.02 mL of standard with unknown sample. The ferrozine assay was used for both soluble and total Fe(II) concentrations.
Samples were pulled for sulfate concentrations at the same sample time that was taken for Fe(II) concentrations. Samples were injected into a microcentrifuge tube, and centrifuged at maximum speed for 5 minutes. Next, 0.10 mL of supernatant was taken for analysis. Sulfate samples were diluted with Milli-Q water and dispensed into 0.50 mL vials. Samples were quantified by ion chromatography on a Dionex 100 system fitted with an AS4A column with conductivity detection (Dionex Corp., Sunnyvale, CA, USA)
(Senko et al., 2008).
Amplex Red glucose assay kits (Invitrogen, Carlsbad, CA) were used for measuring glucose concentration. 10 µL of sample was taken for glucose assay. Samples were diluted in an Amplex Red reaction buffer. 100 µL of diluted samples were injected into wells of a 96-well plate. Glucose standards were made for 0 µM, 10 µM, 25 µM, 50
µM, and 100 µM. Samples were incubated for 30 minutes at room temperature and away from light. Wells were then read using a microplate reader (Spectra Max Plus 384,
Molecular Devices) to analyze results at 560 absorbance.
To measure pH, 0.2 µL of sample was injected into a fresh microtube. Microtubes were vortexed. pH was measured using a micro pH sensor. Samples were taken from cultures once a week for pH measurement in the third round of cultures.
Samples were collected for scanning electron microscopy (SEM). Samples were fixed in anoxic 2% glutaraldehyde. Fixed samples were dehydrated in filter paper, in an ethanol series. Dehydration was done to reach critical point drying. Dehydrated samples were Au coated for observation. Two scanning electron microscopes were used: a Hitachi
14 S-3500 and a Hitachi 4500. An accelerating voltage of 20 kV was used for a range of 0.5 to 10 µm.
DNA from each enrichment culture was taken for microbial community analysis to determine if different types of organisms are enriched under oxic and anoxic conditions. Fe(III) hydroxides were removed from samples by 0.3 M ammonium oxalate washing (Nicomrat et al.,2006; Senko et al., 2008; Brantner et al., 2014)
DNA was extracted using MoBio (MoBio Laboratories, Inc., Carlesbad, CA)
Powerbiofilm DNA isolation kits, according to the manufacturer’s instructions. Qubit 3
Fluorometer was used to test for DNA. 16S rRNA genes were sequenced using the
Illumina platform. Illumina data was processed by Molecular Research LP, MR DNA.
Diversity assay bTEFAP Illumina 16S rRNA sequencing was done. Sequence libraries were evaluated, including taxonomic assignments, in the QIIME environment (Caporaso et al., 2011).
15 CHAPTER III
RESULTS
3.1 Approach
An Fe(III) reducing bacteria medium was created with Fe(III) as a terminal electron acceptor, using Fe2(SO4)3 as the source for Fe(III) phases. Glucose was the electron donor for Fe(III) reduction. Cultures were inoculated with iron mound sediment from the Mushroom Farm to enrich Fe(III) reducing bacteria. At the start of a round, cultures were transferred to fresh media (Figure 3, arrows). Cultures were established under anoxic and oxic conditions. Oxic conditions were used to examine the influence of oxygen on Fe(III) reducing bacteria. Oxygen was replenished to ensure that anoxic conditions did not form within the oxic cultures. Oxic cultures were made using atmospheric air, 20% oxygen, and replenished every 30 days. The Fe(II) concentration was measured to follow the trend of Fe(III) reduction. Fe(II) concentrations increased once a transfer occurred for a new round of inoculated media (Figure 3).
16 Figure 3.
17 Figure 3. Fe(II) concentrations, sulfate concentrations, glucose concentrations, and pH values of the anoxic and oxic cultures over 159 day period. Solid orange diamonds represent oxic cultures. Solid blue squares represent the anoxic cultures. Yellow circles represent the oxic cultures replenished with oxygen. Purple circles represent the anoxic culture incubated with an oxic culture of the previous round. Green triangles represent the uninoculated media bottles (A) Fe(II) concentration. The solid symbols represent total Fe(II) concentrations and the open symbols represent soluble concentrations. (B) The sulfate concentration. (C) Testing the difference between the availability of oxygen to different cultures. (D) Glucose concentrations. (E) pH. The arrows indicate when enrichments were transferred into fresh media.
18 3.2 First Round (Figure 3, Days 0-72)
In the initial enrichment, the Fe(II) concentration reached a maximum concentration of 9.14 mM in oxic conditions, and a maximum concentration of 21.76 mM in anoxic conditions. The maximum concentration was reached in 36 days in anoxic conditions and 18 days in oxic conditions. Oxic cultures were maintained for 72 days, 36 days more than the anoxic cultures (Figure 3A). The goal in prolonging the oxic cultures was to ensure that no further Fe(III) reduction would occur. The majority of oxic Fe(III) reduction occurred over the first 10 days (Figure 3A). In the subsequent transfers, the oxic culture produced more Fe(II) and it approached the maximum concentration of the anoxic culture. Within the first 10 days of the incubation, oxic Fe(III) reduction was comparable to anoxic Fe(III) reduction. The rate of Fe(III) reduction in oxic conditions in the first 10 days was 0.73 mM of Fe(II)/day. The rate of Fe(III) reduction in anoxic conditions in the first 10 days was 0.65 mM of Fe(II)/day. The rate of Fe(III) reduction, about 0.70 mM of Fe(II)/day, was the same between oxic and anoxic conditions in the first 10 days (Figure 3A). The bacteria in the first incubation, that were capable of Fe(III) reduction in oxic conditions, survived in the subsequent cultures. Fe(III) reducing bacteria capable of Fe(III) reduction in oxic conditions would continue to live. Bacteria capable of reducing either oxygen or Fe(III) in this medium would be capable of living on in oxic conditions. The oxic Fe(III) reducing bacteria would likely become the majority of the bacteria remaining in oxic cultures. The soluble Fe(II) for the round follows the same pattern as the total Fe(II) concentrations. Soluble Fe(II) composes most of the total Fe(II) concentration (Figure 3A). The dissolution of Fe(III) through Fe(III) reduction results in the soluble Fe(II) (Zinder et al., 1986). Fe(II) would adsorb onto
19 Fe(III) at higher pH. As pH increases, adsorption of Fe(II) and Fe(III) increases (Nagh et al., 2005). Fe(II) adsorption is greater close to pH of 5.0 (Nagh et al., 2005). Soluble
Fe(II) is the issue that causes concern for understanding the influence of oxygen on
Fe(III) reduction.
The concentration of the media bottles averaged 0.32 mM, ± 0.07, mM of Fe(II), before being inoculated. Fe(II) concentration did not change in the uninoculated media bottles. Because Fe(II) did not increase in the uninoculated media bottles, Fe(III) reduction in this experiment can be determined as a biotic process. Additionally, Fe(II) concentration levels can be used to determine the activity of Fe(III) reducing bacteria in anoxic and oxic conditions.
Regarding terminal electron acceptors, sulfate reduction is thermodynamically the least favorable compared to oxygen and Fe(III) (Table 1). High concentrations of Fe, Mn,
2- and SO4 contaminate AMD streams (Tarutis et al., 1992). Fe concentrations in AMD streams are higher than Mn concentrations (Tarutis et al., 1992). Fe and Mn reduction both occur in AMD streams (Tarutis et al., 1992). Mn reduction is known to already occur in AMD, therefore MnO2 (Table 1) was not added into the medium used for this experiment. Mn reduction has a similar potential to oxygen (Table 1), therefore if Fe(III) reduction is tested for in oxygen, it would work similarly in the presence of Mn. When sulfate reduction occurs, the decrease in sulfate concentration indicates that the more favorable terminal electron acceptors are not present. Sulfate reduction only occurs when
Fe(III) and oxygen are unavailable in the cultures, because both Fe(III) and oxygen reduction are more thermodynamically favorable than sulfate reduction. The sulfate concentration data followed the similar pattern to the uninoculated controls (Figure 3B).
20 The uninoculated patterns can be used as a reference for the concentration without sulfate reduction occurring (Figure 3B). Standard deviation indicates that there was not a change between the uninoculated bottles and the cultures. Therefore, there was no evidence supporting sulfate reduction in either anoxic or oxic cultures.
3.3 Second Round (Figure 3, Days 72-124)
After the first transfer into fresh media, the second round of cultures exhibited similar extents of Fe(III) reduction between anoxic and oxic conditions. Oxygen did not inhibit Fe(III) reduction during the second round. Production of Fe(II) was 0.50 mM/day in both oxic and anoxic conditions (Figure 3.A). Fe(II) stopped being produced 29 days after the culture was transferred into fresh media, for both oxic and anoxic cultures
(Figure 3A). The maximum concentration of Fe(II) was 17.16 mM of Fe(II) production after 36 days in anoxic conditions. The maximum concentration of Fe(II) in oxic conditions was 13.99 mM. The anoxic cultures were observed an additional 16 days longer than the oxic cultures. The anoxic cultures were extended to establish that the maximum concentration of Fe(II) would not increase to a closer maximum concentration similar to the amount of Fe(II) reached in the first round, anoxic cultures (Figure 3A).
The sulfate concentrations did not vary between anoxic conditions, oxic conditions, and the uninoculated (Figure3B). There was no evidence of sulfate reduction in any condition.
21 3.4 Third Round (Figure 3, Days 124-159)
The Fe(III) reduction trend in the third round for anoxic and oxic conditions were similar to the previous rounds. Oxygen did not inhibit Fe(III) reduction in the oxic cultures. In the third round, oxic Fe(III) reduction reached a maximum concentration of
13.71 mM of Fe(II) 31 days after the culture transfer (Figure 3A). The anoxic Fe(II) production reached a maximum concentration of 17.73 mM 28 days after the culture transfer (Figure 3A). When considering the standard deviation, the Fe(II) production peaks of oxic and anoxic cultures were similar.
During the third round, an additional experiment was conducted to further assess the influence of oxygen on Fe(III) reduction (Figure 3C). There was a finite amount of oxygen available in every oxic culture. Oxygen reduction was possibly depleting oxygen faster than it was being replaced after 30 days. If oxygen was depleted completely, the oxic cultures would become anoxic. The addition of the culture replenished with oxygen would confirm that bacteria were capable of reducing Fe(III) in oxic conditions, regardless of oxygen concentrations. By continually adding oxygen to the culture, the culture would be guaranteed to have both the Fe(III) hydroxide and oxygen during the entire length of the experiment. The inoculum for these cultures was from the oxic cultures of the first transfer. The oxic culture was inoculated with a culture from the previous oxic culture and injected with air at the time of the transfer into fresh media.
The culture replenished with oxygen was inoculated with a culture from an oxic medium and injected with air every day samples were taken. The purpose of replenished oxygen was to see if the repetitive addition of oxygen would affect the amount of Fe(III)
22 reduction. If oxygen is continually replenished, then oxygen will continue to be an available terminal electron acceptor. By guaranteeing oxygen remains available to the aerobes, Fe(III) would not become the most favorable terminal electron acceptor available during the experiment. In the previous two rounds, oxic and anoxic Fe(III) reduction yielded similar Fe(II) production values. When oxic testing was over 30 days, more oxygen was added. One of the cultures was an anoxic culture incubated with an oxic culture of the previous round. The anoxic culture incubated with an oxic culture was established to observe if a change in the cultures’ atmosphere from oxic to anoxic would have any change on the production of Fe(II). Using an enrichment from oxic conditions for the second transfer into an anoxic culture would demonstrate if the bacteria were able to reduce Fe(III) in both anoxic and oxic conditions. If the anoxic culture that was incubated with an oxic culture produced Fe(II), then the Fe(III) reducing bacteria were capable of Fe(III) reduction under anoxic and oxic conditions. If the culture did not produce Fe(II), then the specific Fe(III) reducing bacteria from the oxic enrichment were only capable of reducing Fe(III) in the presence of oxygen.
The maximum concentration of Fe(II) in the oxic culture was 13.71 mM, 31 days after inoculation (Figure 3A). The rate of Fe(II) production in oxic conditions was 0.83 mol of Fe(II)/day, during the first 14 days. The culture replenished with oxygen reached a maximum concentration of 11.23 mM Fe(II), 19 days after inoculation (Figure 3C). The rate of Fe(II) production in the culture replenished with oxygen was 0.71 mol of
Fe(II)/day, during the first 14 days. The culture replenished with oxygen had a decrease in Fe(II) concentration between days 145-159, which was 21 days after the cultures were transferred. The culture replenished with oxygen had a depletion of Fe(II) concentration
23 (Figure 3A and C). Fe(II) depletion was not represented in any other cultures (Figure 3).
The replenishment of oxygen throughout the incubation did not inhibit Fe(III) reduction.
However, the additional oxygen did change the length at which Fe(III) reducing bacteria are capable of reducing Fe(III) in oxic conditions. The Fe(II) decreased in the culture replenished with oxygen because the availability of both oxygen and Fe(II) would mean that Fe(II) oxidation could occur (Eq. (2)). Indicated by the Fe(II) depletion (Figure 3A),
Fe(II) oxidation does occur in the culture that was replenished with oxygen. Fe(II) oxidizing bacteria were present in the culture. The bacteria reducing Fe(III) could also be oxidizing Fe(II).
The anoxic culture that was inoculated from an oxic culture produced the greatest
Fe(II) concentrations compared to the other third round cultures (Figure 3C). The rate of
Fe(II) production in the anoxic culture that was inoculated from an oxic culture was 1.0 mol of Fe(II)/day, during the first 14 days. The bacteria present, that were actively reducing Fe(III) in oxic conditions in the previous round, displayed more Fe(III) reduction in anoxic conditions. Fe(II) production in both conditions indicates that the bacteria found specifically in the anoxic culture that was inoculated from an oxic culture are capable of Fe(III) reduction in either condition. The bacteria of the anoxic culture that was inoculated from an oxic can reduce Fe(III) in both anoxic and oxic conditions. The
Fe(II) production of this culture indicates that some bacteria are capable of Fe(III) reduction in both anoxic and oxic conditions, within acidic settings.
Glucose levels were measured in the third round for the oxic culture and the anoxic culture (Figure 3D). The required electron donor would come from glucose in the media. The changes in glucose concentrations would be due to glucose depletion during
24 metabolism. The measured glucose level was about 4.11 mM for day 0. The glucose was depleted over 35 days. At the end of 35 days, oxic cultures had about 1.57 mM of glucose remaining, anoxic cultures had about 2.54 mM of glucose remaining. Uninoculated cultures remained around 4.10 mM of glucose during the 35 day period. Glucose depletion can be linked to microbial activity because glucose in the uninoculated cultures did not change, whereas the inoculated cultures lost 2.43 mM in oxic conditions and 1.53 mM in anoxic conditions (Figure 3D). The trend indicates that oxic cultures used more glucose than anoxic cultures. Fe(III) reducing bacteria would use either Fe(III) or oxygen as a terminal electron acceptor, depending on availability in the experiment. The overall glucose levels indicate that there was more microbial activity in oxic conditions. In anoxic conditions, the only electron acceptor is the Fe(III). In the oxic conditions, there are two available electron acceptors: Fe(III) and oxygen. Therefore, the bacteria can reduce either Fe(III) or oxygen. Because there are Fe(III) reducing bacteria in the oxic cultures, there was more abundance of terminal electron acceptors, the glucose concentration should be more depleted in oxic cultures. The glucose concentrations were more depleted in the oxic conditions (Figure 3D). The decrease in glucose levels in both oxic and anoxic conditions is evidence of respiration occurring in these cultures. The electron donor, glucose, decreases while the product of the terminal electron acceptor,
Fe(II), was increasing (Figures 3A, C, and D).
The pH for the anoxic and oxic cultures was recorded during the third round
(Figure 3E). In both oxic and anoxic conditions, pH increased then decreased. (Figure
3E). The pH needed to remain between 2.5-4.5 for the range of pH of AMD streams
(Cravotta et al., 1999). The pH began at 2.5 at the start of inoculations (Figure 3E). After
25 the experiment began with a pH of 2.5, the pH stayed above 3.0 and below 5.0 for 28 days. The pH is within the right conditions for acidophilic Fe(III) reducing bacteria. The only time the pH was below 3.0 was at the start of the experiment, and in the uninoculated media. The change in pH is linked to biotic activity because there was no change in pH within the uninoculated media bottles. Fe(III) reduction consumes H+ (Eq.
(7)). Therefore, when Fe(III) reduction happens in acidic conditions and will increase the pH. The pH will continue to increase during Fe(III) reduction until Fe(III) or glucose is depleted. The pH was changed due to the biotic activity of Fe(III) reducing bacteria.
3.5 Scanning Electron Microscopy
Samples were examined under a scanning electron microscope (SEM) (Figures
4-8). SEM was performed to observe the bacteria from different cultures. Bacteria found in the SEM confirms the bacterial growth in the oxic and anoxic cultures.
26 Figure 4. SEM of the uninoculated medium that contains Fe(III) hydroxide particles.
27 Figure 5. SEM of an oxic culture. (A) Bacterium attached to Fe(III) hydroxides. This bacterium is found within a heavy concentration of Fe(III) hydroxides. (B) Bacterium with patches of Fe(III) hydroxides attached at one end. (C) Bacterium that is not attached to a high concentration of Fe(III) hydroxide. (D) Bacterium that is attached to Fe(III) hydroxides.
28 Figure 6. SEM of an oxic culture. (A) Bacterium attached to a large cluster of Fe(III) hydroxides on the left side. (B) Bacteria that appear to be separating. (C) The left bacterium of Figure 6B. The connection between these two bacteria is highlighted with the blue arrow. The bacterium appears to be separating from the other, split between different patches of concentrated Fe(III) hydroxides. (D) The right bacterium of Figure 6B. The bacterium is attached to Fe(III) hydroxides along most of the rod-shaped cell.
29 Figure 7. SEM of an anoxic culture. (A) Rod-shaped bacterium with appendages on either end. The bacterium does not appear physically attached to Fe(III) hydroxides. (B) Rod-shaped bacterium with an appendage on one end. Bacterium not attached to Fe(III) hydroxides. (C) Several bacteria located around concentrations of Fe(III)hydroxides. Two of the bacteria on the bottom left appear to be attached to the same Fe(III) hydroxides. There is one larger bacterium to the top. Two bacteria are close in the bottom right. (D) These two bacteria are on the bottom right of Figure 7C. The left bacterium is almost completely covered in the Fe(III) hydroxide.
30 Figure 8. SEM of a culture in a medium consisting of glucose, but no Fe(III). More bacterium was seen from this Fe(III)-free culture than any of the others. (A) Bacteria attached to each other, not visibly attached to Fe(III) hydroxide particles. (B) Three bacteria overlapping each other. (C) Three bacteria, none are attached to one another. (D) A bacterium in the Fe(III)-free medium attached to particles on one side of the rod shape.
31 Several bacteria could be seen in an oxic culture (Figures 4 and 5) interacting with the
Fe(III) hydroxides. The bacteria in oxic cultures were attached to the Fe(III) hydroxides
(Figure 5A). Certain Fe(III) particles were mainly attached to the bacterium at the ends
(Figure 5C). Not all Fe(III) phases were located on the bacterium’s ends, some Fe(III) hydroxides were along the elongated surface of that bacterium instead (Figure 5D). A bacterium from an oxic culture was found fully encased in Fe(III) hydroxides (Figure
6A). Two bacteria were seen attached to Fe(III) hydroxide particles and the other bacterium (Figure 6B-D). Rod-shaped bacteria were also found in the anoxic cultures
(Figure 7A-B). Bacteria found in anoxic conditions were larger in both size and quantity.
Additional bacteria were found that looked like those of the oxic cultures, and comparably more bacteria were visible under SEM from anoxic cultures (Figure 7C). At higher magnification, the bacterium can be seen interacting with the Fe(III) particles
(Figure 7D).
The last culture examined with SEM was an oxic culture that did not contain
Fe(III) in the medium (Figure 8). The oxic culture without Fe(III) was inoculated with an oxic culture from round 2. If there was bacteria present in the oxic culture without
Fe(III), then the bacteria’s presence indicate the bacteria from the oxic culture without
Fe(III) are capable of reducing both Fe(III) and oxygen. Bacteria found in the culture without Fe(III) appeared similar to the bacteria in the cultures containing Fe(III) hydroxides (Figure 8). The bacteria found in the Fe(III) hydroxide rich cultures could be reducing Fe(III) or oxygen. The bacteria were found in the Fe(III)-free culture are visually similar in size and shape to the bacteria found in the oxic cultures with Fe(III).
According to the SEM, bacteria were more abundant in this Fe(III)-free culture than any
32 other culture that was incubated in a medium containing Fe(III) (Figure 4-8). Bacteria found in Fe(III)-rich anoxic and oxic cultures were scarce in comparison to the culture without Fe(III) hydroxides. The Fe(III)-free culture had several clusters of bacteria
(Figure 8A-B). The oxic cultures established in Fe(III) hydroxides grew in the presence of both terminal electron acceptors. There was Fe(III) and oxygen reduction occurring in the oxic Fe(III) hydroxide cultures (Figures 3A and D). However, more bacteria were visible in SEM of the oxic cultures without Fe(III) hydroxides.
While SEM offers an opportunity to look at the bacteria that were visible within each culture, SEM does not provide an accurate representation of the cell abundance of the enrichment cultures. There are noticeable differences between the bacteria within the different cultures. Most of the bacteria in oxic conditions were about 1-3 µm long.
Bacteria in the anoxic cultures were larger than that of the oxic cultures. The largest anaerobic bacteria were over 10 µm (Figure 7A), other anaerobic bacteria were 2-5 µm long (Figure 7C-E). The larger bacteria (Figure 7A and B) are likely different bacteria than the ones that are more abundant and similarly visual to the bacteria found in oxic cultures (Figure 5, 6, 7B and C). The SEM with the most bacteria visible was the Fe(III)- free, oxic cultures (Figure 8). In oxic cultures, there were both Fe(III) reducing bacteria and aerobic oxygen reducing bacteria. Additionally, Fe(III) reducing bacteria present may also use other terminal electron acceptors. The cultures with the Fe(III) hydroxides would contain Fe(III) reducing bacteria that can also reduce oxygen. The anaerobic bacteria were more abundant in SEM than that of the aerobic bacteria SEM. A group of bacteria was found together in the anoxic culture (Figure 7C), while most of the bacteria found in the oxic cultures were seperated, except for two bacteria physically linked together
33 (Figure 6C). Even though the size and quantity of bacteria in oxic conditions are not as apparently visible compared to the anoxic conditions, bacteria are still present in oxic conditions (Figure 6).
3.6 Taxonomic Composition of Cultures
16S rRNA sequence was obtained for cultures from the first and third rounds
(Figue 9). The samples used for rRNA data was obtained from the cultures at the end of each round. The taxonomic level being observed is by class. The first round was mostly composed of Bacilli in both the anoxic culture, 49%, and the oxic culture, 76% (Figure
9). Bacilli was the majority class of bacteria found in every culture except the third-round anoxic culture (Figure 9). The third-round anoxic bacteria was predominantly Clostridia,
96% (Figure 9). Firmicutes was the dominant phylum bacteria in all the cultures.
Firmicutes has been known to reduce Fe(III) in iron ore caves (Parker et al., 2017).
Firmicutes is also recorded in cultures observed from southeastern Ohio surface mining sources (Poncelet et al., 2014).
The next most abundant bacteria predominantly in the oxic cultures is
Gammaproteobacteria (Figure 9). The first-round anoxic and oxic cultures contained 2% and 12%, respectively, of Gammaproteobacteria (Figure 9). The third-round anoxic culture and oxic culture replenished with oxygen contained 1% and 8%, respectively, of
Gammaproteobacteria (Figure 9). The comparison between the oxic culture and oxic culture replenished with oxygen indicates that Gammaproteobacteria is most active in oxic conditions with the most oxygen. The idea that Gammaproteobacteria lives in
34 cultures with the most oxygen is confirmed because the third-round anoxic culture had
1% and the anoxic culture incubated with an oxic culture had 1% (Figure 9). However the most abundant bacteria in the anoxic culture incubated with an oxic culture was Bacilli, the bacteria mainly found in the oxic cultures. Therefore, the Bacilli were capable of reducing Fe(III) in anoxic and oxic conditions.
The experiments proved that bacteria were able to reduce Fe(III) in both oxic and anoxic conditions. Fe(II) concentration increased in both oxic and anoxic cultures (Figure
3) The different experiments in round 3 showed variation between oxic and anoxic
Fe(III) reduction (Figure 3C). While oxygen did not inhibit Fe(III) reduction, it did accelerate Fe(II) oxidation (Figure 3C), indicated by the depletion in Fe(II). Fe(II) even slightly declined in the oxic cultures of round 1, when the culture was given extra oxygen after 30 days (Figure 3A). Due to comparison of the Fe(II) concentrations (Figure 3) to the bacteria abundances (Figure 9), Bacilli and Gammaproteobacteria are known bacteria that can reduce Fe(III) in oxic conditions.
35 Figure 9. 16S rRNA gene abundance of sequences at the class-level.
36 CHAPTER IV
DISCUSSION
Oxygen is the more thermodynamically favorable terminal electron acceptor compared to Fe(III). Fe(III) reduction is one of the most impactful processes in anoxic environments, because of organic degradation (Coleman et al., 1993) and the production of dissolved Fe(II) (Cravotta et al., 1999). Due to thermodynamic favorability (Table 1),
Fe(III) reduction should not occur in the presence of oxygen, thereby inhibiting Fe(III) reduction. Previous AMD related studies (Brantner et al., 2014; Burwick et al., 2017) have reported Fe(III) reduction occurring in oxygen. This experiment focused on the influence of oxygen on Fe(III) reduction and the enrichment of Fe(III) reducing bacteria.
I hypothesized Fe(III) reducing bacteria would continue to metabolize in both anoxic and oxic settings, under acidic conditions. Fe(III) reduction was occurring in the presence of oxygen without being inhibited.
Fe(III) Reduction in AMD
Anoxic zones in AMD are the locations of biotic Fe(III) reduction (Senko et al.,
2008). Microbial Fe(III) reduction influences many processes: the oxidation of organic matter, the release of phosphate and trace metals into water supplies, and the release of high concentrations of Fe(II) into ground waters (Lovley et al., 1993). The areas with the most Fe(III) reduction will have an increase in Fe(II) production (Senko et al., 2008).
Fe(III) phases are reduced at different rates. Fe(III) reduction is more energetically favorable at a low pH (Burwick et al., 2017), (Figure 3). Fe(III) hydroxides are reduced
37 much more rapidly than Fe(III) crystalline structures (Kostka et al., 2002). Fe(III) reduction rates are affected by the Fe(III) reducing bacteria (Kostka et al., 2002). Acidic bacteria reduced Fe(III) during this experiment (Figure 3).
Biotic Fe(III) reduction was indicated to be inhibited by oxygen (Lovley et al.,
1993). Oxygen is toxic for certain Fe(III) reducing bacteria (Lovley et al., 1993).
However, Fe(III) reduction has been observed occurring in the presence of oxygen
(Burwick et al., 2017). Although schwertmannite, a Fe(III) hydroxide phase, cores that were incubated with Mushroom Farm sediment showed the most Fe(III) reduction in deeper, anoxic deposits, Fe(III) reduction did occur in oxic conditions as well (Burwick et al., 2017). The Fe(III) reducing bacteria were still slightly inhibited by oxygen
(Burwick et al., 2017). The majority of AMD Fe(III) reduction is happening in deeper, anoxic deposits within iron mounds. However, this experiment has proven that the upper oxic zones of AMD stream will have Fe(III) reducing bacteria, not inhibited by oxygen.
Due to the high abundance of Fe(III) reducing bacteria detected in AMD iron mounds, Fe(III) reducing bacteria have access to the organic carbon and Fe(III) needed for Fe(III) reduction (Brantner et al., 2014). Fe(III) reduction is one possible respiratory
2- substrate. Other electron acceptors include oxygen, MnO2, SO4 , CO2 (Table 1). AMD is predominantly contaminated by Fe(III) hydroxides (Bertel, 2011). Sulfate is another electron acceptor present in AMD, but sulfate is a less thermodynamically favorable electron acceptor than Fe(III) (Table 1). Mn is a contaminant in AMD streams as well
(Tarutis et al., 1992). Fe concentrations are higher than Mn concentrations in AMD streams (Tarutis et al., 1992).
38 Brantner et al. (2014) studied depth-dependence of geochemical and microbiological processes. Oxygen was present within the first 2 cm of sediment
(Brantner et al., 2014). With increasing depth, dissolved Fe(II) increased as oxygen was depleted (Brantner et al., 2014). Fe(II) oxidation in the greater depths of iron mounds results in higher Fe(III) concentrations and depleted oxygen (Eq. (2)). Fe(III) reduction would be a thermodynamically favorable process (Table 1) after the accumulation of
Fe(III) and depletion of oxygen (Eq. (7)). Sequentially, Fe(III) reduction would then cause increased Fe(II) concentrations in greater depths of sediment, describing the process of AMD iron mounds (Brantner et al., 2014). The production of Fe(II), due to
Fe(III) reduction, will continue until organic carbon and Fe(III) are depleted. Fe(II) oxidation will not happen again unless in the presence of oxygen.
Understanding the depletion of oxygen in AMD systems due to Fe(III) reducing within iron mounds was important in understanding how oxygen depletion would occur in this experiment. Evident due to Brantner et al. (2014), Fe(II) oxidizing bacteria will deplete oxygen with increased depth. Additionally, oxygen diffuses vertically into the sediment and is consumed by bacteria that respire oxygen (Brantner et al., 2014).
Fe(III) reducing bacteria used glucose, provided in the medium, for an electron donor. The Fe(III) reducing bacteria are chemoorganoheterotrophs (Willey et al., 2009).
Aerobes can use glucose as an electron donor with oxygen as the terminal electron acceptor (Eq. (5)). In oxic conditions, there are Fe(III) reducing bacteria and oxygen reducing bacteria using glucose. In anoxic conditions, there is only evidence of Fe(III) reducing bacteria using glucose. Glucose could be depleted by both oxygen reduction and
Fe(III) reduction, resulting in the glucose depletion being greatest in oxic cultures
39 compared to anoxic cultures (Figure 3D). However, if both glucose decreases and Fe(II) increases, then a link between Fe(II) production can be made to Fe(III) reducing bacteria using glucose as an electron donor and Fe(III) as a terminal electron acceptor (Eq (7)).
With recent research indicating the possibility of Fe(III) reduction in oxic conditions (Brantner et al., 2014; Burwick et al., 2017), this experiment proves that
Fe(III)-reducing bacteria do respire Fe(III) in oxic conditions. When comparing all three rounds, the first round of testing was the only round that more Fe(III) was reduced in anoxic conditions than in oxic conditions. In the two subsequent rounds, Fe(III) reduction was not restricted by the presence of oxygen.
The decrease in Fe(II) in the oxic culture replenished with oxygen means Fe(II) oxidation is occurring (Eq. (2)). The change in Fe(II) concentration in the culture replenished with oxygen indicates that the produced Fe(II) from Fe(III) reducing bacteria in oxic conditions will be immediately oxidized by Fe(II) oxidizing bacteria. Fe(II) oxidation will replenish Fe(III) in the system, which will lead to more Fe(III) reduction.
If there is still oxygen available, Fe(II) oxidation will occur again. In 35 days, the culture reached 11.23 mM Fe(II) and decreased to 4.57 mM Fe(II) (Figure 3C). No other culture throughout the experiment went that low in Fe(II) concentration. In an AMD system, if bacteria can live in oxic conditions within AMD streams (Brantner et al., 2014; Burwick et al., 2017), Fe(III) reduction will occur in the entire system, but Fe(II) oxidation will only occur in oxic conditions. While soluble Fe(II) is being oxidized in oxic conditions,
Fe(II) concentrations will remain unchanged in anoxic conditions. The Fe(III) hydroxides will continue to be produced in an iron mound system due to Fe(II) oxidation, and will be followed by Fe(III) reduction, in acidic settings. Fe(III) reduction in iron mounds will
40 continue to produce Fe(II). Iron mounds are the location of a cycle between Fe(II) oxidation and Fe(III) reduction. Oxygen is a main part of Fe(II) oxidation and does not inhibit Fe(III) reduction.
AMD Sulfate Reduction
Sulfate is the least thermodynamically favorable terminal electron acceptor compared to oxygen and Fe(III) (Table 1). Oxygen is the most favorable terminal electron acceptor (1.23 volts), followed by Fe(III) (1.06 volts), then sulfate (0.25 volts)
(Table 1). Based off thermodynamic favorability, sulfate reduction should only occur in anoxic conditions, when Fe(III) is unavailable. Sulfate reduction in AMD streams would result in sulfide production (Burton et al., 2008).
In the experiment, sulfate concentration was measured to identify if there was sulfate reducing bacteria present having an effect on Fe(III) reduction. If sulfate concentrations decreased, than sulfate reduction occurred, indicating the thermodynamic favorability of sulfate at the time. Sulfate reducing bacteria will produce H2S, which will reduce Fe(III) hydroxides to form iron sulfides (Coleman et al., 1993). When sulfides are produced in anoxic marine settings, sulfate reducing bacteria will produce FeS2 through
Fe(III) reduction (Berner et al., 1983). FeS2 production from sulfate reducing bacteria would be represented by an increase in Fe(II), and a decrease in sulfate concentration. If
Fe(II) production matches sulfate depletion at the same time, then there would be evidence of sulfate reducing bacteria cause Fe(III) reduction.
41 The concentration of sulfate does not change throughout the experiment, when considering standard deviation of anoxic and oxic cultures, and the uninoculated media.
Changes in Fe(II) concentrations within anoxic conditions do not match with any significant changes in the sulfate concentrations (Figure 3A and B). Fe(III) reduction was not occurring due to sulfate reducing bacteria producing Fe(II). The significance of sulfate reduction not occurring reaffirms that Fe(III) reduction in the experiment is due to
Fe(III) reducing bacteria.
Change in pH
Fe(III) reduction is more thermodynamically favorable at low pH (Burwick et al.,
2017). Fe(III) reduction is known to be favorable in acidic conditions, because H+ ions are depleted in the process (Eq. (7)). Circumneutral pH ranges from 6.5-7.5. Fe(III) reduction is favorable under acidic conditions of pH less than 6.5 (Bethke et al., 2011).
Coalmine discharge has an average pH of 2.5 - 4.5 (Cravotta et al., 1999). The pH will increase during Fe(III) reduction, because the H+ ions are depleted in Fe(III) reduction.
The medium for the experiment was created at a pH of 4.5. The pH was at 2.5 for the start of the experiment (Figure 3E), after autoclaving. The acidic pH values of the experiment are favorable for Fe(III) reducing bacteria conditions, in either anoxic or oxic conditions.
The bacteria did not live solely in acidophilic bacteria conditions of less than 3.0
(Johnson and McGinness, 1991) during the duration of the experiment (Figure 3E). The pH within the experiment remained within the pH range of AMD streams, pH of 2.5 - 4.5
42 (Cravotta et al., 1999). The Fe(III) reducing bacteria that were active in the anoxic and oxic cultures were capable of reducing Fe(III) in acidic conditions higher than the strict pH of environments, pH less than 3.0, measured for acidophilic bacteria. The experiment had pH results indicative of Fe(III) reducing bacteria, because of the increase in pH once the experiment began.
Bacteria found using a Scanning Electron Microscope (SEM)
The SEM offered detailed images of interactions between the bacteria and the
Fe(III) phases. Rod shaped bacteria were observed in the inoculated anoxic and inoculated oxic samples. All of the bacteria viewed under the scanning electron microscope were rod shaped. Rod shaped Fe(III) reducing bacteria have been isolated before (Finneran et al., 2003). Rod shaped bacteria that reduced Fe(III) through glucose oxidation have been previously isolated (Küsel et al., 1999). Fe(III) reduction was observed within a pH range of 2.1-5.8, where the optimum pH was 3.2 (Küsel et al.,
1999). The average pH during the experiment was 3.24 in oxic conditions and 3.63 in anoxic conditions. The pH of the experiment was in range for the conditions of the Fe(III) reducing bacteria referenced by Kusel et al. (1999).
When considering Fe(III) reducing bacteria in AMD streams, the Fe(III) reducing bacteria must be capable of living in acidic conditions. Johson and McGinnes (1991) classified acidophilic, heterotrophic Fe(III) reducing bacteria in the presence oxygen within the Acidiphilium. Acidiphilium is a genus positioned in the phylum of
43 Proteobacteria (Sievers et al., 1994). Acidophilic bacteria that were rod shaped match the shape of bacteria that were found within the cultures.
Taxonomic Composition of Cultures
Proteobacteria is one of the phylum of which the acidic Fe(III) reducing bacteria in this experiment are from because it is capable of living in the pH range that was measured in the experiment. Certain Fe(III) reducing bacteria within Proteobacteria are strictly anaerobic bacteria (Lonergan et al., 1996). Fe(III) reducing Proteobacteria
Gammaproteobacteria have been found within Fe(III) reduction zones in anoxic conditions (Oswald et al., 2016). Testing has been done to predict dissimilatory metal reduction activity of Gammaproteobacteria (Wee et al., 2013). Wee et al. (2013) only demonstrated Gammaproteobacteria ability to reduce Fe(III) and Mn(IV) in anaerobic conditions. Gammaproteobacteria activity was used as an indicator for anoxic conditions
(Oswald et al., 2016). However, Gammaproteobacteria was the second most predominant bacteria in oxic conditions of both the first and third rounds of the experiment (Figure 9).
Gammaproteobacteria was not very abundant in the anoxic cultures (Figure 9). Oswald et al. (2016) observed Gammaproteobacteria methane oxidation in oxic/anoxic transition zone. Gammaproteobacteria has previously been observed in oxic conditions (Oswald et al., 2016) and anoxic conditions (Wee et al., 2013), but only Fe(III) reduction in anoxic conditions. This experiment specifically demonstrates Gammaproteobacteria ability to reduce Fe(III) in oxic conditions.
44 Firmicutes was the most abundant bacteria phylum in all of the cultures (Figure
9). The most abundant Firmicutes in anoxic conditions was Clostridia. Clostridia is a strict anaerobic bacteria capable of Fe(III) reduction (Park et al., 2001). Clostridia was the most abundant in the anoxic cultures (Figure 9), reaffirming that this class of
Firmicutes is anaerobic. The most abundant Firmicutes in oxic conditions was the Bacilli
(Figure 9). Bacilli has been observed reducing Fe(III) prior, but as a strict anaerobic bacteria (Boone et al., 1995). Kanso et al. (2002) tested Bacilli growth in both anaerobic and aerobic conditions and found the bacteria was only able to successfully reduce Fe(III) and Mn(IV) in anaerobic conditions. The Bacilli strain that strictly reduced Fe(III) anaerobically, did so in a pH range of 7-8 (Kanso et al., 2002). The pH of the experiment was within 2.5-4.5 (Figure 3E). Bacilli was capable of Fe(III) reduction in oxic conditions (Figure 3A, Figure 9) when the pH was acidic, below 7 (Figure 3E).
Iron Mound Applications
The Fe(III) hydroxides precipitate and deposit along the surface of AMD streams
(Senko et al., 2008). The importance of Fe(III) at AMD streams is due to the ability to remove it, while soluble Fe(II) cannot be physically separated from the stream.
Limestone beds and limestone channels are used in passive AMD remediation (Cravotta and Trahan, 1999). Limestone remediation removes dissolved metals like Fe(III), Al3+,
2+ and Mn from streams (Cravotta and Trahan, 1999). Limestone, CaCO3, reacts with
Fe(II) and water, resulting in Fe(III) phase (Bologo et al., 2008). Fe(III) is physically separated from AMD to limit the amount of Fe(III) hydroxides that attach to the limestone surfaces and clogging limestone beds (Senko et al., 2008). Keeping the
45 limestone beds and channels clean keeps the AMD remediation continuous by neutralizing the pH of AMD waters and removing Fe (III) hydroxides (Cravotta and
Trahan, 1999).
If bacteria can reduce Fe(III) in oxic conditions, then bacteria may be reducing
Fe(III) from iron mounds at oxic locations on AMD sites. Fe(III) hydroxides that precipitate over limestone beds could be a continuous source for Fe(III) reduction in oxic conditions. Fe(III) reduction occurring in oxic conditions will reverse the process that happens due to AMD remediation. This experiment proved that Fe(III) reduction does occur in the presence of oxygen (Figure 3A). Therefore, Fe(III) reducing bacteria could be actively undoing AMD remediation that depends on the precipitation of Fe(III) hydroxides. Iron mounds left alone will have a build up of Fe(III) hydroxides. Attempting to depend on iron mounds without the assistance of limestone will lead to a continuation of Fe(II) oxidation and Fe(III) reduction. Limestone will neutralize the pH, and Fe(III) reducing bacteria will be unable to produce Fe(II). The dependence on neutral pH indicates the limitations of iron mounds being left unchecked. Iron mounds left unattended will continue to have Fe(III) reduction and Fe(II) oxidation occurring in the system because the pH will remain acidic, allowing Fe(III) reducing bacteria to continue.
46 CHAPTER V
CONCLUSION
AMD is an issue that impacts eastern and southern Ohio currently. Mining exposes FeS2 to oxygen and results in iron oxidation, and followed by low pH. Passive remediation techniques, such as limestone beds, are in place to help neutralize the acidic streams that are between 2.5-4.5 pH. AMD remediation sites remove soluble metals from
AMD contaminated waters. In the remediation process, soluble Fe(II) is oxidized to
Fe(III). Fe(III) hydroxides can be physically removed from AMD contaminated water.
Unfortunately, Fe(III) reducing bacteria in AMD streams work against remediation.
Fe(III) reducing bacteria continue to contaminate AMD streams by increasing the concentration of soluble Fe(II). AMD impacted streams are polluted with Fe(III) hydroxides. Fe(III) reduction occurs in AMD streams when bacteria use Fe(III) as the terminal electron acceptor. According to thermodynamic favorability, bacteria will respire using whatever terminal electron acceptor provides the most energy for work.
Fe(III) reducing bacteria are known to be active in deeper deposition at AMD streams, due to low oxygen levels. Fe(III) reduction is an anaerobic process. However, evidence of
Fe(III) reducing bacteria in oxic conditions indicates that iron being precipitated as
Fe(III) hydroxides can still end with recontamination of Fe(II) in AMD streams. While
Fe(III) hydroxides are actively removed, Fe(III) reduction is also taking place. The water is still being contaminated with soluble Fe(II). This process was known to be occurring in the anoxic zones of AMD, but it is also occurring in the oxic zones. While Fe(III)
47 reduction occurred in oxic zones, it was unclear the impact that oxygen was having on
Fe(III) reducing bacteria.
Cultures were incubated from Mushroom Farm, a known AMD contamination site in North Lima, Ohio. Oxic cultures were created to observe if oxygen inhibits Fe(III) reducing bacteria. Anoxic cultures were created to compare and observe the anaerobic process. Media was left uninoculated to determine if Fe(III) reduction was a biological process. Due to no change in Fe(II) concentration in the uninoculated media bottles,
Fe(III) reduction was reaffirmed to be a biotic process. Fe(II) and sulfate concentrations were measured over 159 day period, divided into three rounds. Glucose and pH were measured in the third round. The Fe(II) concentration increased in both anoxic and oxic cultures after every transfer into fresh media. Fe(III) reducing bacteria successfully reduced Fe(III) in oxic conditions. Fe(III) reduction was not inhibited by the presence of oxygen when comparing the Fe(II) production in both oxic and anoxic conditions.
However, oxygen does have an effect on the long term process of oxic Fe(III) reduction.
Fe(II) concentrations produced in oxic conditions will begin going through Fe(II) oxidation. Therefore, while oxygen does not inhibit Fe(III) reduction, it did cause Fe(II) to deplete. The Fe(II) concentrations found in AMD streams will mostly remain in the anoxic conditions.
The taxonomy of 16S rRNA abundances in the experiment indicates how not all
Fe(III) reducing bacteria are capable of Fe(III) reduction in oxic conditions. Bacilli and
Gammaproteobacteria were active in oxic conditions, while Clostridia was most active in anoxic conditions. Bacilli was previously seen only active in anaerobic conditions, but under circumneutral pH. Therefore, there are two understandings of whether oxygen will
48 inhibit Fe(III) reduction: not all Fe(III) reducing bacteria can reduce Fe(III) in the presence of oxygen and some bacteria can only reduce Fe(III) in the presence of oxygen in acidic conditions.
There are multiple microbial processes in AMD streams: aerobic oxygen reduction, Fe(II) oxidation, and Fe(III) reduction. When there is a thermodynamically favorable electron donor and terminal electron acceptor, microbial electron transfers will occur. Fe(III) reducing bacteria may reverse the precipitation of iron out of AMD streams. Fe(III) reduction is not inhibited by oxygen. Fe(III) reducing bacteria are going to be active in both oxic and anoxic zones of AMD streams. Because limestone remediation neutralizes pH, limestone beds will prevent Fe(III) reduction in oxic conditions. However, iron mounds will still have an acidic pH. Iron mounds are limited by Fe(III) hydroxide production. Unless Fe(III) reducing bacteria are removed or accounted for in the process, soluble Fe(II) will continue to be redistributed back into
AMD streams. The cycle of Fe(II) oxidation and Fe(III) reduction will continue in oxic conditions, in acidic settings.
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