MICROBIAL COMMUNITY COMPOSITION AND ACTIVITIES IN WET FLUE GAS
DESULFURIZATION SYSTEMS
A Thesis
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Gregory D. Martin
May, 2017 MICROBIAL COMMUNITY COMPOSITION AND ACTIVITIES IN WET FLUE GAS
DESULFURIZATION SYSTEMS
Gregory D. Martin
Thesis
Approved: Accepted:
______Advisor Dean of the College Dr. John M. Senko Dr. John Green
______Faculty Reader Dean of the Graduate School Dr. Teresa J. Cutright Dr. Chand Midha
______Faculty Reader Date Dr. Hazel A. Barton
______Department Chair Dr. Stephen C. Weeks
ii ABSTRACT
This project was conducted to characterize microbial communities and slurry in wet flue gas desulfurization (wFGD) units at coal burning power plants. An additional objective of this research was to ascertain microbial activity and the potential for microbial mercury metabolism. Coal fired power plants in the U.S. alone are responsible for emitting over 50 tons per year of Hg0 into the atmosphere.
A consequence of this microbially produced MeHg is increased toxicity with distance in food webs, eventually reaching humans where it can damage nervous systems and impair fetal development. Therefore, Hg bioaccumulation as a direct result of increased anthropogenic Hg0 emissions is a global concern. To address the chemistry and microbial activities of wFGD slurry, I determined the physiochemisty of three wFGD systems. I then quantified the activity of microorganisms in the wFGD slurry using live/dead cell counts, respirometry experiments monitoring O2 consumption over time, a Hg reduction experiment monitoring total Hg loss over time, and total RNA sequencing reads. Microbial community composition was established by evaluation of 16S rRNA gene sequences recovered from the systems.
I found live cells and increased aerobic respiration in live slurry incubations when compared to deactivated slurry samples. When comparing the 16S rRNA gene sequencing data, the wFGD communities all possessed lower relative abundances than the more physiochemically moderate river ecosystems, which provide the
iii source water. However, they differed between sites in physiochemisty and predominant genera recovered through DNA analysis, as well as RNA sequencing and culturability. Two of the sites contained high percentages of the thermophilic taxa, Hydrogenophilaceae Hydrogenophilus and Hydrogenophilaceae Thiobacillus.
This research strongly indicates microbial activity in the physiochemically extreme wFGD environment and suggests that microorganisms introduced from the moderate source water can adapt to the more extreme wFGD environment.
Research goals of this thesis
a. Determine the general physiochemisty and microbial communities of
wFGD slurry.
b. Establish the presence of active microbial communities in wFGD and
elucidate the potential for these active communities to contribute to
mercury reduction in wFGD slurry.
iv ACKNOWLEDGMENTS
I would like to acknowledge my advisor, Dr. John Senko for his guidance and patience over the last two years. His unflagging support and thoughtful insight into experimental design, data analysis and interpretation were invaluable. I would also like to thank Dr. Teresa Cutright, Dr. Hazel Barton, Tom Quick, Anne Marie Hartwell,
Robert Miller, Shagun Sharma, Ceth Parker, and Olivia Hershey for their insight, support, and technical expertise, and Mark Golightly for his assistance in procuring samples and expertise in all aspects of wFGD system chemistry and operation. I would like to thank the Electric Power Research Institute (EPRI) for so generously supporting my research. Finally I would like to thank Dr. Francisco Moore for encouraging me to pursue a graduate degree, as well as the many friends and colleagues who have assisted me along the way.
v TABLES OF CONTENTS
Page
LIST OF FIGURES ...... ix
LIST OF TABLES ...... xi
CHAPTER
I. INTRODUCTION AND BACKGROUND ...... 1
Core Concept ...... 1
Significance of Coal Burning in Global Mercury Cycles ...... 2
Overview of Flue Gas Desulfurization Units ...... 3
wFGD Chemistry ...... 6
Significance of Coal Combustion in Mercury Speciation ...... 7
Significance of Environmental Mercury ...... 10
Environmental Mercury Speciation ...... 10
Mercury Emission Control Methods ...... 11
wFGD Mercury Speciation ...... 11
Microorganisms in wFGDs ...... 12
Microbial Mercury Metabolism ...... 14
Mercury Reemission and Retention ...... 15
Research Questions ...... 16
vi Hypotheses ...... 17
Approach ...... 17
II. MATERIALS AND METHODS ...... 19
Site Descriptions and Characterizations ...... 19
Sample Collection Protocol ...... 19
Experimental Design ...... 20
Chemical Characterization of wFGD Slurry and Source Water ...... 20
Microbial Enumerations ...... 21
wFGD Slurry Incubations: Oxygen Consumption Experiment ...... 21
wFGD Slurry Incubations: Mercury Loss Experiment ...... 22
Nucleic Acid-Based Microbial Community Characterization ...... 22
III. RESULTS ...... 26
Chemical Characteristics of wFGD Slurries and Source Water ...... 26
Microbial Enumerations ...... 30
wFGD Slurry Incubations: Oxygen Consumption Experiment ...... 32
wFGD Slurry Incubations: Mercury Loss Experiment ...... 38
Nucleic Acid-Based Microbial Community Characterization ...... 41
16S rRNA gene transcript characterization ...... 58
IV. DISCUSSION ...... 63
Microbial Adaptability to Extreme Environments ...... 64
Implications of Findings ...... 69
wFGD Microbial Mercury Metabolism ...... 70
vii Future Directions ...... 72
V. CONCLUSION ...... 74
REFERENCES ...... 75
APPENDIX ...... 82
viii LIST OF FIGURES
Figure Page
1. Schematic of a typical wFGD system, with stars indicating microorganism sources (adapted from Brown et al., 2012) ...... 5
2. Illustration of the Hg cycle (adapted from Utah DEQ, 2016)...... 9
3. Oxygen consumption by Shewanella oneidensis, with panels A-D at 20oC, 40oC, 55oC, and 70oC, respectively. Blue = S. oneidensis. Red = empty (air only) chambers...... 33
4. Oxygen consumption by microorganisms associated with site S slurry, with panels A-D at 20oC, 40oC, 55oC, and 70oC, respectively. Blue = active samples, Red = deactivated samples...... 35
5. Oxygen consumption by microorganisms associated with site M slurry, with panels E-H at 20oC, 40oC, 55oC, and 70oC, respectively. Blue = active samples, Red = deactivated samples...... 36
6. Oxygen consumption by microorganisms associated with site P slurry, with panels I-L at 20oC, 40oC, 55oC, and 70oC, respectively. Blue = active samples, Red = deactivated samples...... 37
7. Total Hg concentrations (µmol/L) over a 38-day period. Dark blue = site S active, Red = site S deactivated, Green = site M active, Purple = site M deactivated, Teal = site P active, Orange = site P deactivated...... 40
8. Graph of rarefaction analysis for wFGD systems from both sampling trips and source water. Dark blue = S1, red = M1, green = P1, purple = S2, teal = M2, orange = P2, light blue = SW, and pink = PW...... 46
9. Phylum-level (class-level for Proteobacteria) OTU relative abundances for bacterial 16S rRNA genes. Frame A includes chloroplast-attributable sequences (Cyanobacteria) for all samples. Panel B includes chloroplast-attributable sequences (Cyanobacteria) in source water samples only...... 50
ix 10. PCoA analysis of wFGD associated communities and source water samples. A-C use weighted metrics. D-F use unweighted metrics. A = PC1 vs. PC2, B = PC1 vs. PC3, C = PC2 vs. PC3, D = PC1 vs. PC2, E = PC1 vs. PC3, F = PC2 vs. PC3 ...... 53
11. PCoA analysis of wFGD associated communities and source water samples with chloroplast-attributable sequences (Cyanobacteria) removed. G-I use weighted metrics. J-L use unweighted metrics. G = PC1 vs. PC2, H = PC1 vs. PC3, I = PC2 vs. PC3, J = PC1 vs. PC2, K = PC1 vs. PC3, L = PC2 vs. PC3...... 54
12. Genera-level of all OTUs from bacterial 16S rRNA genes found above 5% in wFGD and source water samples. All genera under 5% grouped and represented by top orange ban ...... 57
x LIST OF TABLES
Table Page
1. Aqueous chemistry of wFGD slurry and source water (river) samples for first and second sampling trip (Mark Golightly, personal communication). Below detection level = BDL. Not measured = NM...... 28
2. wFGD slurry cell counts (cells/mL slurry) by site, using live/dead staining and fluorescent microscopy...... 31
3. DNA concentrations by site and sampling trip...... 42
4. Estimates of microbial abundances and diversity of wFGD systems...... 44
5. Total and perfectly aligned reads produced by de novo assembly of total RNA from wFGD sample sites...... 60
xi CHAPTER I
INTRODUCTION AND BACKGROUND
Core Concept
Wet flue gas desulfurization systems (wFGD) are the primary method for SOx,
NOx, and some Hg removal from coal combustion exhaust. These systems operate at
2- high temperatures with elevated dissolved Ca, Mg, SO4 , and Cl concentrations.
Since active microbial communities have been found in physiochemically extreme environments, as varied as arctic ice and deep-sea hydrothermal vents, it is likely that microorganisms are present and active within wFGD systems. Identifying and determining the activities of the microorganisms present within wFGD systems could lead to enhanced retention of Hg, thus limiting the environmental damage caused by that element. Limiting Hg emissions from coal-fired power plants is necessary, but increasingly difficult with current physical and chemical means.
Addressing possible microbiological activities of wFGD systems is increasingly important. Especially since bioaccumulation of methylmercury (MeHg) in the environment, as a direct result of burning coal, is a major health and environmental concern. Therefore, establishing the presence and activities of the microorganisms in wFGDs is an important first step.
1 Significance of Coal Burning in Global Mercury Cycles
Industrialized nations largely rely on coal burning power plants for electricity generation. In the US, 33% of electricity is furnished by coal burning power plants (USEIA, 2016). The energy yield, as well as the quantity and identity of toxins and trace pollutants found in coal vary by coal grade and origin (Bowen and
Irwin, 2008). The four coal grades, listed in order of decreasing energetic yield are: anthracite, bituminous, sub-bituminous, and lignite. These grades correspond to the geological age and composition of the coal. Lignite contains the most water, has the poorest energy yields, and is the youngest; anthracite has the lowest water content, highest energy yields, and is the most rare coal. Coal sources in the eastern U.S. are primarily bituminous, while coal reserves in the western U.S. are mostly sub- bituminous (USEIA, 2016). West Virginia, Kentucky, and Pennsylvania are three of the top five coal producing states, and account for 12%, 8%, and 5% of the nation’s coal, respectively, as of 2013 (USEIA, 2016). In 2015, the U.S. had an estimated 478 billion short tons in demonstrated reserve base of coal so it is unlikely focus will shift away from coal as a viable energy source (USEIA, 2016).
Coal formed when shallow oceans flooded large vegetated areas. The salt seas evaporated leaving the sulfur, mercury, nitrogen, and other trace metals now associated with coal. These sulfur compounds, nitrogen-oxygen compounds, and trace metals resulted primarily from anaerobic bacterial activity in the peat. The plant matter was transformed into peat, which was then overlain by soil. With time, heat, and pressure, coal formed. Coal-forming plants are the main contributors to
2 low sulfur coals, and lower rank coals tend to have higher amounts of thermally unstable organic sulfur types as a result of microbial metabolism (Calkins, 1994).
Coal burning power plants remain one of the single largest contributors to air pollution, especially: SO2, NOx, Hg/MeHg, and other trace metals (USEPA, 2016).
Burning coal releases sulfur, most notably in the form of SO2, the primary culprit for acid rain. Acid rain is the result of SO2 gas emissions reacting in the atmosphere to form sulfuric acid which lowers the pH of rain and causes the acidification of soils and aquatic systems (Likens et al., 1996; Srivastava and Jozewicz, 2001; Likens et al.,
1979). Hg, in the form of Hg0, is also released into the atmosphere during coal combustion due to the high temperatures. It is therefore important to develop and implement strategies for pollution control. Exhaust scrubbers, called flue gas desulfurization units (FGDs), were introduced starting in the 1970s to combat the acid rain problem (Córdoba, 2015).
Overview of Flue Gas Desulfurization Units
FGD units are large systems designed for SO2, NOx, and, to a lesser extent,
Hg2+ removal. There are two broad categories of FGD systems, wet (wFGD) and dry
(dFGD), which is misleading because both types use water. wFGD systems use much more water and are the most common system type in the U.S. and globally, due to the relative simplicity of the design (Nolan, 2000). Many wFGD systems in the U.S. were installed as a response to emission regulations created by the Clean Air Act
Amendments (CAAA) of 1990, and as a result of being retrofits, come in an array of sizes and configurations. However, they all follow the same general layout and have similar chemistry. Exhaust from the combustion burners (flue gas) is passed into the
3 FGD tank above the liquid slurry level, and wetted by spray nozzles with a water and limestone or slaked lime mixture. The scrubbed gas continues through the top of the system and exits the stack to the atmosphere. The reacted limestone slurry falls to the liquid portion of the tank and continues to react forming CaSO42H2O (gypsum), with the addition of O2 (also called forced oxidation) (Córdoba, 2015). The bottom slurry mixture is cycled out of the tank for processing. The solids are dewatered and disposed of or sold for industrial uses such as wallboard, cement, and soil augmentation to offset operational costs. The liquids are treated and released back to the source lake or river, or recycled into the wFGD system as makeup waters with the addition of fresh limestone. Figure 1 illustrates the major components of a typical wFGD system.
4
Figure 1. Schematic of a typical wFGD system, with stars indicating microorganism sources (adapted from Brown et al., 2012)
5 wFGD Chemistry
The upper portion of the wFGD tank, encompassing the area between the limestone slurry sprayers and the slurry surface can be referred to as the gas-to- liquid contact zone, and when operating between pH 5-6, produces the following series of reactions (Córdoba 2015). The dissolution and hydrolysis of the SO2 gas is accompanied by the disassociation of sulfurous acid and limestone dissolution. After bicarbonate acid-base neutralization and equilibrium desorption of CO2, we arrive at equation 1.
Equation 1
CaCO + SO +2H O →CaSO ⋅2H O +CO 3(s) 2( g) 2 (aq) 3 2 (aq) 2( g)
The lower half to two-thirds of the wFGD tank contains the liquid slurry composed of the unreacted limestone, water, ash, and the products of the first series of reactions carried out in the gas-to-liquid contact zone. The slurry in the reaction tank continues the reactions of the gas-to-liquid zone, proceeding to form
2- CaSO42H2O, as a result of the longer residence time provided to oxidize SO3 to
2- SO4 as shown in equation 2.
Equation 2
1 CaSO3 ⋅2H2O(aq) + O2( g) →CaSO4 ⋅2H2O(s) 2
At pH of 4.5-5.5, the abundance of protons results in increased sulfuric acid and lower gypsum yields, relative to less acidic systems. Additional limestone is added to replace the reacted calcium carbonate and raise the pH to near neutral,
6 which yields gypsum and CO2 as the final products. SO2 and O2 concentrations, and the pH of the limestone slurry determine the ratio of CaSO30.5H2O (calcium sulfite dihydrate) to CaSO42H2O formation in the slurry. 50-60% calcium sulfate to gypsum ratios are normal when no additional O2 is fed into the system (natural oxidation), as compared to 90% gypsum when additional O2 is introduced into the wFGD (forced oxidation). The chemistry of any given wFGD system is well characterized and tightly controlled by plant operators because proper system chemistry is essential to optimal pollutant removal and operational costs
(Poullikkas, 2015). This is of interest from a microbial viability viewpoint because the physiochemical stability of a wFGD system should allow for the establishment of microbial communities.
Significance of Coal Combustion in Mercury Speciation
Almost all Hg in coal burned in the furnaces of power plants, regardless of original speciation, is converted to Hg0 because the high operating temperatures exceed Hg vapor point (Lee et al., 2006; Kolker, Senior, and Quick, 2006). Hg0 is insoluble, extremely volatile, and easily transported globally once released to the atmosphere. Of the trace metals found in coal, Hg is a major concern due to the rise in methylmercury (MeHg) from microbial activity and its affects on the biosphere.
Anthropogenic activities have increased the amount of Hg participating in the global cycle and available for methylation by releasing Hg from storage in the lithosphere
(Driscoll et al., 2013). Through 1990, municipal waste combustors, medical waste incinerators, and coal burning power plants supplied two-thirds of total atmospheric Hg emissions, each contributing over 50 tons per year. As of 2005, only
7 power plants continue to emit large amounts of Hg, over 50% of the total U.S air pollution (USEPA, 2016).
Figure 2 is a simplified depiction of the major parts of the Hg cycle as it relates to air pollution, human toxicity and Hg speciation. Starting from the left, anthropogenic release of Hg from the lithosphere is the primary means of introducing new Hg to the global Hg cycle. Once in the released into the environment, large bodies of water are the major contributors to atmospheric Hg, which is then reduced through photoreactions and redeposited. The most concerning aspect of the Hg cycle from a human health aspect are the methylating processes that occur in anoxic water and sediments. As shown in the lower portion of Figure 2, a miniature Hg cycle driven by microorganisms produces the MeHg that travels through food webs and eventually reaches human consumers. Since it is incredibly difficult to control Hg speciation and methylation in the environment, it is advantageous to focus efforts on anthropogenic emission sources.
8
Figure 2. Illustration of the Hg cycle (adapted from Utah DEQ, 2016).
9 Significance of Environmental Mercury
Humanity has attempted to use Hg as medicine for millennia while simultaneously recognizing its detrimental health effects (Ozuah, 2000; Swiderski,
2008). Although all Hg species are toxic to humans, we now know that toxicity is dependent on the Hg speciation at exposure and the severity and longevity of the exposure (Ozuah, 2000; Swiderski, 2008; Gochfeld, 2003). Exposure in adults results in nerve damage, sensory impairment, numbness, spasticity, dementia, and a host of other maladies; in infants, especially in utero, the results can include mental retardation, deafness, blindness, microcephaly, and cerebral palsy (Ozuah, 2000).
Therefore, it is valuable to human health to understand how Hg behaves in the environment, in addition to knowing how it arrives there.
Environmental Mercury Speciation
The three most common Hg species are elemental mercury (Hg0); inorganic
Hg or mercurial salts (Hg+/Hg2+); and organic, methylated mercury (MeHg) (Ozuah,
2000). MeHg is the most toxic and most widely distributed species in biota, owing to its propensity for bioaccumulation (Gochfeld, 2003). This is facilitated in part by the lipid solubility of Hg (Watras and Bloom, 1992). MeHg accumulates in the cytoplasm of phytoplankton which is then integrated into the biomass of higher trophic levels, while inorganic Hg accumulates in cell membranes which are more readily excreted by consumers (Mason et al., 1994). This distinction is significant, as it accounts for the bioaccumulation of MeHg into higher trophic levels, most notably large fish and humans.
10 Inorganic Hg is converted to MeHg in natural systems via microbial activity in water logged or aquatic sediments and to a lesser extent in algal mats (Gochfeld,
2003; Ozuah, 2000). Only 1-10% of Hg in sediments is methylated, but 90-99% of
Hg found in biota is MeHg, which raises the concern for human exposure (King et al.,
2000). Hence, it is important to control anthropogenic Hg releases into the environment.
Mercury Emission Control Methods
To date, there is no single treatment option capable of removing all air pollutants from coal exhaust. Instead, multiple technologies are implemented in series, the order of which can impact toxin removal effectiveness (Córdoba, 2015;
Ochoa-Gonzalez, Diaz-Somoano, and Martinez-Tarazona, 2014). Generally this treatment train includes an electrostatic precipitator (ESP) or a fabric filter (FF) and or selective catalytic reduction (SCR) coupled with either wet or dry FGD units
(USEPA, 2016). An ESP or FF removes much of the ash and significant oxidized Hg, with up to 99% removal efficiency of particle bound Hg (Hgp) (Zhang et al., 2016).
2+ SCR systems are employed for NOx and SO2 removal, but can also increase Hg capture by 34-85% (Zheng et al., 2014; Zhang et al., 2016).
wFGD Mercury Speciation
Although wFGD systems have successfully reduced SO2 and NOx emissions from coal combustion, they have only partially reduced Hg emissions, with 98% SO2 and 60-95% Hg2+ removal efficiencies possible (Córdoba, 2015; Kolker, Senior, and
Quick, 2006; Zhang et al., 2016). wFGDs achieve variable total Hg removal, between
11 30-85%, because Hg is most soluble in its oxidized form, but enters the treatment train in its reduced form (Hg0) (Schuetze et al., 2012). Hg emissions may be further compounded by reemissions, the process by which soluble Hg2+, which has been captured in the slurry portion of a wFGD unit, escapes from the exhaust stack either because of reduction of Hg2+ to Hg0 or binding of Hg2+ to small particulate matter
(Pavlish et al., 2003). Reemission can take the form of Hg2+ bound to fine particulates, or chemical and microbial activity in the slurry portion through reduction of Hg2+ to Hg0 (Córdoba, 2015; Ochoa-Gonzalez, Diaz-Somoano, and
Martinez-Tarazona, 2014).
Hg found in flue gas primarily exists as elemental (Hg0), oxidized (Hg2+) and particle bound (Hgp) Hg (Meij and te Winkel, 2006). Studies have shown a complex range of factors affect Hg speciation in wFGDs, including physiochemical characteristics of the slurry such as ash, chloride concentration, pH, and oxygen concentrations (Córdoba, 2015). Although microorganisms play vital roles in Hg cycling in other environments, very little work has been conducted on the impacts microbial communities may have on Hg reemission or retention in the slurry portion of these wFGD units (Brown et al., 2012). It is possible that microbial populations in the wFGD units are capable of oxidizing Hg0 to Hg2+, thereby limiting reemissions; reducing Hg2+ to Hg0 and methylating Hg2+ to MeHg, thus increasing retention rates; or that both processes occur simultaneously.
Microorganisms in wFGDs
Microorganisms use enzymes and thermodynamic disequilibrium to leverage energy. They accomplish this through redox reactions, the process of stripping
12 electrons from one compound and transferring them to another. The transfer of electrons allows for the generation of adenosine triphosphate (ATP), which is the energetic “currency” of the cell. Although microorganisms are incredibly abundant and diverse throughout all earth systems, some systems are physiochemically challenging for life. These extreme systems include deep ocean geothermal vents and hot springs, because these systems are hot, often deprived of sunlight and other nutrients, and have high sulfur concentrations (Jannasch and Mottl, 1985; Pace,
1997; Jørgensen et al., 1992). These conditions make microbial growth difficult, and the resulting microbial communities are often significantly less complex than more moderate systems.
High temperatures, in the 50-60oC range, induced by the exhaust, and high levels of Ca2+, Mg2+, SO2-, ash, metal ions, and gypsum, unreacted limestone, and pH from 5-6.5 all make wFGDs harsh environments (Córdoba, 2015). The rivers or lakes that provide source water and the crushed limestone come from more moderate systems and provide the microorganisms that may populate wFGDs. Some wFGDs use forced oxidation to achieve higher gypsum yields making the reaction tank an oxic system. Other systems use magnesium lime and are designed to limit the amount of O2 entering the system (inhibited oxidation), resulting in an more anoxic slurry environment (Córdoba, 2015; Shen et al., 2013; Srivastava and
Jozewicz, 2001). Any active microorganisms found in these systems would need the genetic capability and metabolic pathways to enable them to establish residence in such an extreme environment.
13 Microbial Mercury Metabolism
Many aerobic bacteria are able to control Hg toxicity from environmental exposure by volatizing Hg through the reduction of Hg2+ and Hg-compounds to Hg0
(Figueiredo et al., 2014). Hg reduction and demethylation capabilities are associated with a group of genes located in the mer operon, which is found in a broad range of aerobic bacteria (Figueiredo et al., 2016). Aerobic microorganisms with these gene groups posses one of two gene suites, narrow spectrum or broad spectrum
(Figueiredo et al., 2016). The narrow spectrum gene suite only enables the reduction of Hg2+ to Hg0, while the broad spectrum gene suite encodes for the reduction of Hg2+ and the demethylation of Hg (Figueiredo et al., 2016). In one study, conducted with oxic and anoxic lake sediments, oxic sediment samples methylated Hg more slowly and had lower averaged cell counts than anoxic samples; however, oxic and anoxic sediments had equal Hg volatilization rates, while the inactivated control sediments had no Hg volatilization (Regnell and
Tunlid, 1991).
In anoxic, subsurface environments, Hg0 can be oxidized to Hg2+ by nitrate- reducing, iron-reducing, sulfate-reducing, and/or fermentative bacteria (Colombo et al., 2013). Hg0 oxidation has also been found in oxic systems, and the presence of cellular biomass may increase Hg2+ retention (Holm and Cox, 1975). Hg2+ may also be methylated in sediments, a process initially attributed to sulfate reducing bacterial (SRB) and later Fe(III)-reducing bacteria (King et al., 2000; Podar et al.,
2015). Through the identification hgcA and hgcB, two genes necessary for Hg methylation, many more Hg methylators have been found, including syntrophic
14 Deltaproteobacteria, Firmicutes, and Archaea (Podar et al., 2015). The identification of gene families implicated in microbially mediated Hg transformation is important as it enables the identification of a diverse range of Hg oxidizing, reducing, methylating, and demethylating microorganisms possible. The immense diversity in microorganisms capable of Hg metabolism makes it likely that there are species present in wFGD units contributing to Hg speciation within the system.
Based on the extreme physiochemistry of wFGD systems, similar environments include geothermal vents, acid mine drainage (AMD)-impacted systems, and sulfur rich ocean sediments. Geothermal systems and hot springs are the most similar chemically and should have similar microbial communities (Hoaki et al., 1994; Burton and Norris, 2000; Eloe-Fadrosh et al., 2016). Depending on the redox status of the system, expected active microorganisms could be either aerobic or anaerobic, although the physical shape of a particular wFGD tank, may create both oxic and anoxic zones within the same system. Due to the high temperatures, all anticipated species would be thermophilic. Within a wFGD system, microbially mediated oxidation of Hg0 to Hg2+ could contribute to increased total Hg capture and lower Hg emission rates. Likewise, microbial reduction of Hg2+ to Hg0 could lead to decreased total Hg capture and overall higher Hg emission rates due to volatilization.
Mercury Reemission and Retention
Hg not captured in wFGD units is released into the atmosphere as Hg0, a portion of which is photochemically oxidized to Hg2+, deposited, and eventually integrated into sediments, where is can be methylated (Selin 2009). Hg2+ in wFGD
15 unit slurries can be reemitted to the environment through absorption to coal ash, and by chemical and biological reduction to volatile Hg0 (Ochoa-Gonzalez, Diaz-
Somoano, and Martinez-Tarazona, 2014; Córdoba, 2015). The fate of Hg captured in wFGDs is not fully understood, but it can be found in the synthetic gypsum used in wallboard manufacturing. A study conducted by Kairies et al. (2006) found Hg levels between 140-1500 μg/kg dry weight in both FGD-gypsum and finished wallboard
(Kairies et al., 2006). They also found that the Hg tended to associate with slower settling Fe-rich portions of the slurry, indicative of sorption to the Fe phases. In general, Hg speciation in wFGD units is highly dependent on unit physiochemistry,
- especially the concentrations of SO2, HCl, and pH which interact to form Cl Hg-S species, effectively inhibiting Hg reemission (Córdoba, 2015).
Research Questions
MeHg as a result of increased global Hg0 emissions is a serious concern because of its detrimental health effects in humans. Coal fired power plants remain major contributors of atmospheric Hg0, despite the implementation of air pollution control devices, including wFGD units. These wFGD units are physiochemically extreme environments, which pose challenges to microbial growth and influence Hg speciation within the system. However, the known diversity of Hg metabolizing species in other systems, and the successful sequencing of DNA recovered from wFGDs make it likely that microorganisms are involved in Hg speciation within wFGD units (Hsu-Kim et al., 2013).
Before the role of microorganisms in wFGD speciation can be addressed, the activity and identities of the microbial communities present must be established. To
16 address the microbial communities present and active in the slurry, the habitat of the wFGD needs to be determined. This will enable wFGD slurry comparison to the source water and systems where microbial community structures and functions are known, as well as more effective culturing techniques. Identifying the microorganisms present, in conjunction with characterizing the wFGD slurry will allow for more effective evaluation of potentially active microorganisms.
Therefore, I propose the following research objectives:
1. Determine the general physiochemisty and microbial communities
of wFGD slurry.
2. Establish the presence of active microbial communities in wFGD
and elucidate the potential for these active communities to
contribute to mercury reduction in wFGDs.
Hypotheses
H1. The extreme physiochemistry of wFGD slurries will be reflected in
microbial communities more similar to extreme natural systems, like
geothermal settings.
H2. Live microbial communities will be present and active in wFGD slurry
and will have Hg metabolizing capabilities.
Approach
To better understand the specific environments of wFGD systems in relation to other physiochemically extreme environments, and to establish the physiochemical context for the microbial communities, I recorded pH and quantified
17 2+ 2+ 2+ 2- 2- - dissolved Mg , Ca , Fe , Cl , SO4 , and NO3 concentrations for each site. To determine if there are active microorganisms in wGFDs, as apposed to dead or inactive cells, I employed several approaches. First, to look for and estimate the abundance of live microorganisms in my samples, I conducted live/dead staining and cell counts. To ascertain aerobic activity I measured O2 loss over time. To assess microbial Hg reduction in the slurry samples, I conducted an Hg experiment monitoring total Hg loss in active samples compared to deactivated controls, over time.
To ascertain which microorganisms are not only present, but also active, I conducted nucleic acid-based community characterization. I extracted DNA and sequenced partial bacterial 16s rRNA genes so that I could determine community composition of the microorganisms in my wFGD systems. Lastly I used total rRNA sequencing to see which microorganisms are most active based on the relative abundances of sequenced rRNA copies. This was the final step to answering my research question concerning which microorganisms are active, and not just present, in wFGD slurry.
18 CHAPTER II
MATERIALS AND METHODS
Site Descriptions and Characterizations
All three sample sites are located at coal fired power plants located along the
Ohio River, and all operate multiple wFGD units with the river as their source water.
The wFGD units are uniform within the sites, but vary in volume and operation between sites. The temperature of all the wFGDs is maintained near 55oC. The first site, S, is a 2,233 megawatt (WM) plant that relies on limestone wFGD units with forced oxidation (FirstEnergy Corp., 2016). The second site, M, is a 2,490 MW plant that uses a magnesium lime mixture in its wFGDs, along with inhibited oxidation
(FirstEnergy Corp., 2016). The third site, P, is a 1,300 MW plant that operates a modified magnesium lime system with higher MgOH levels (FirstEnergy Corp.,
2016). Additionally, Site P has square, rather than the more typical, cylindrical, wFGD tanks.
Sample Collection Protocol
Two sets of samples were collected at all three sites, one set August 18th and
19th, 2015 and a second set August 17th and 25rd, 2016. On both sampling trips, site S and site P samples were collected from bleed valves adjacent to the wFGD units.
19 Due to logistics, site M samples were collected from an open flow near the scrubber on the first trip and from a bleed valve adjacent to the wFGD units on the second trip. Each power plant has multiple wFGD units, so samples were collected from the unit recommended by onsite professionals as most representative of overall wFGD operations within the plants. River water samples for 16S rRNA gene analysis were collected on the second sampling trip near the system intake at sites S and P. All samples were collected in sterile containers and transported on ice to The
University of Akron for further analysis. Immediately upon return to the laboratory, for each site, 5 mL of sample was filter sterilized into 5 mL of 0.5 M HCl for atomic absorption spectrometry analysis, 5 mL of sample was filter sterilized in preparation for anion quantification, two 50 mL tubes intended for DNA extraction, and two 50 mL tubes designated for total RNA sequencing were immediately placed in the -700C freezer, all other samples were placed in the refrigerator.
Experimental Design
Chemical Characterization of wFGD Slurry and Source Water
- - 2- Cl , NO3 , and SO4 concentrations were measured with a Dionex ICS-1100
Basic Integrated IC System (Thermo Fisher Scientific Inc., Sunnyvale, CA). Dissolved
Ca, Mg, and Fe2+ concentrations were determined with a Perkin Elmer AAnalyst 700
Atomic Absorption Spectrometer (The PerkinElmer Corp., Norwalk, CT). Each sample pH was determined with a VWR® sympHony™ B10P Benchtop Meter (VWR
Int., Radnor, PA) in lab.
20 Microbial Enumerations
Microbial abundances in wFGD slurries were determined by total live/dead cell counts after staining samples with SYTO® 9 green-fluorescent nucleic acid stain and propidium iodide red-fluorescent nucleic acid stain (Thermo Scientific Inc.,
Waltham, MA) and then viewing with an Olympus BX53F fluorescent light microscope (Olympus Life Science Solutions, Waltham, MA). I determined the volume of slurry each well of a Tekdon 16 well microscope slide (TEKDON, INC.,
Myakka City, FL) held and then counted five grids per well for 5 wells for a total of
25 grids. I used the average of the grid count to estimate the number of both live and dead organisms.
wFGD Slurry Incubations: Oxygen Consumption Experiment
Aerobic microbial activity in slurries was determined by measuring O2 consumption rates using a Micro-Oxymax respirometer (Columbus Instruments,
Columbus, OH) (Poncelet et al., 2014). Test chamber temperatures were maintained using a water bath and deactivated samples were created by autoclaving wFGD slurry. Two active samples and two deactivated, control samples for each site were run at each of the following temperatures: 20oC, 40oC, 55oC and 70oC, to ascertain respiration rates. Additionally, a control using active Shewanella oneidensis in two chambers and air in the other two chambers was run at 20oC to establish baseline activity in active and inactive sampling lines (or channels), as well as instrument noise. Lastly, one chamber with Shewanella oneidensis and one air only chamber were run with each site experiment at each temperature. These incubations allowed
21 me to determine temperature optima for wFGD-associated microorganisms utilizing oxygen.
wFGD Slurry Incubations: Mercury Loss Experiment
Total Hg concentrations in untreated (raw) slurry samples and select samples from the course of a 38-day Hg loss experiment were determined using a
-6 Hydra IIc Hg analyzer with a ≤ 5 μM detection limit (Teledyne Leeman Labs,
Hudson, NH). The experiment was conducted to determine if a side-by-side comparison of active and autoclave deactivated slurry samples would yield different levels of total Hg over time, indicating Hg reduction. The experiment was set up by placing 30 mL of each slurry into separate 100 mL Erlenmeyer flasks and amending the slurry with mercuric chloride to achieve a 100 μM solution. Sites S, M, and P each had two autoclaved deactivated samples and three active incubations. All flasks were incubated at 57oC in a water bath. One mL samples were periodically collected from each flask over 38 days. Samples were collected and total Hg was quantified as described above.
Nucleic Acid-Based Microbial Community Characterization
DNA was extracted using the MoBio PowerBiofilm DNA isolation kit (MoBio
Laboratories, Inc., Carlsbad, CA) from lyophilized slurry solids from wFGD sites collected the first sampling trip, and from centrifuge-pelleted slurry solids collected the second sampling trip. Source water was collected at the wFGD system intakes for Sites S and P on the second sampling trip and filtered onto sterile 2 micron filters and frozen at -70oC until DNA was extracted using the MoBio PowerBiofilms DNA
22 isolation kit. In preparation for Illumina MiSeq based 16S rRNA gene sequencing, I performed PCR and gel electrophoresis to determine if the DNA was amplifiable, and NanoDrop (Thermo Scientific Inc., Waltham, MA), and Qubit 3.0 Fluorometer
(Thermo Scientific Inc., Waltham, MA) to quantify DNA yields.
Extracted DNA from all wFGD and source water samples was shipped to
Molecular Research LP (MR DNA, Shallowater, TX) for Illumina MiSeq, where barcoded 515F, 806R primers were used to amplify partial bacterial 16S rRNA gene through a 28 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) programed for 94°C for 3 minutes, then 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C for 1 minute, and 72°C for 5 minutes final elongation. Multiple samples are pooled in equal proportions based on their molecular weight and DNA concentrations, as determined by a 2% agarose gel. The Illumina DNA library was prepared from calibrated Ampure XP bead purified pooled samples. Molecular
Research LP (www.mrdnalab.com, Shallowater, TX, USA) sequenced the samples on a MiSeq, following the manufacturer’s guidelines, and then processed the sequence data using Molecular Research LP analysis pipeline (MR DNA, Shallowater, TX, USA).
This pipeline joined the sequences, depleted barcodes, removed sequences <150bp, and sequences with ambiguous base calls. Lastly, the sequences were denoised and chimeras removed.
These sequences were then processed in the MacQIIME environment, an open-source bioinformatics pipeline for completing microbiome analysis of raw
DNA sequencing data, using default parameters of QIIME scripts (Caporaso et al.,
2010). While in the MacQIIME environment, I picked operational taxonomic unites
23 based on 97% sequence similarity (OTU0.03) and assigned taxonomic units using the
RDP Classifier 2.2 with the SILVA database (Edgar, 2010; Werner et al., 2012; Wang et al., 2007; Quast et al., 2013). I aligned the OTU sequence reads to the SILVA database using PyNAST and constructed phylogenetic trees using FastTree 2.1.3.
(Caporaso et al., 2010; Quast et al., 2013; Price et al., 2010). I then evaluated the alpha and beta diversities of each system by comparing Shannon indices, phylogenetic distances with weighted and unweighted UniFrac, sampling depth with rarefaction curves, and relative abundances (Lozupone and Knight, 2005).
Unaltered slurry samples from all three sites were shipped on dry ice to Mr.
DNA LAB (Shallowater, TX) for total RNA sequencing. Slurry was centrifuged to concentrate samples and then total RNA was extracted using the MoBio RNA
PowerSoil Total RNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA). DNA contamination was removed using Baseline-ZERO DNase (Epicentre) and then samples were purified using RNA Clean and Concentrator Columns (Zymo
Research). Next, whole transcriptome amplification was conducted using
QuantiTect Whole Transcriptome Kit (Qiagen) and Nextera DNA Sample preparation. Double stranded cDNA concentrations were measured with a Qubit dsDNA HS Assay Kit (Life Technologies), and the diluted samples were fragmented, and adapter sequences and unique indices were added during a 5 cycle PCR. Final library concentrations were measured with a Qubit dsDNA HS Assay Kit (Life
Technologies) and average library size was determined with an Agilent 2100
Bioanalyzer (Agilent Technologies). Libraries were pooled in equimolar ratios of 2 nM. 10 pM of the pooled library was clustered using Illumina cBot and paired end
24 sequenced for 500 cycles on an Illumina HiSeq 2500 system. The returned, unpaired sequence reads were processed using Rockhopper, an open source program for processing bacterial RNA sequence data, and the NCBI database using the BLASTN tool (Altschul et al.,1997). Ribosomes and the rRNA, which compose them, are indicative of the metabolic capabilities of a cell, since more active cells should have more ribosomes.
25 CHATPER III
RESULTS
Chemical Characteristics of wFGD Slurries and Source Water
To better understand the wFGD physical and chemical environments and to compare them with that of the source water, samples were collected from three coal fired power plant wFGD systems during two sampling campaigns. The first sampling campaign was conducted August 18th and 19th of 2015, and the second was carried out August 17th and 25th of 2016. Generally, wFGD systems maintain high temperatures, a pH of 5.5-6, and have relatively high concentrations of dissolved calcium and sulfate within the slurry (Córdoba, 2015).
The average operating temperature for site S was 51oC, site M was 55oC, and site P was 56oC (Mark Golightly, personal communication; Table 1). S and P slurries both maintain lower pH than site M. Site M utilizes magnesium-enhanced lime, which is more soluble and tends to produce more alkaline slurry (Córdoba, 2015).
Within the individual systems, the pH was consistent between the two sampling trips. Site S pH was unavailable from the plant for the first year and 5.44 the following year. Site M pH increased from 6.65 to 6.87. Site P pH decreased from 5.89
26 to 5.82. S and P source water were circumneutral with pH 7.73 and pH 7.75, respectively. wFGD systems maintain relatively stable pH, since pH influences the ratio of calcium sulfate (gypsum) to calcium sulfite in the slurry, and is therefore important to plant operators (Córdoba, 2015). ORP is only monitored for limestone based systems like site S, not inhibited oxidation systems like sites M and P. Site S
ORP was unavailable on the first sampling trip and 172.4 mv on the second (Mark
Golightly, personal communication).
27 Table 1. Aqueous chemistry of wFGD slurry and source water (river) samples for first and second sampling trip (Mark Golightly, personal communication). Below detection level of 5-6 μM = BDL. Not measured = NM.
- Sample Trip pH Temp Cl NO3- SO42- Mg Ca Hg
(oC) (mM) (mM) (mM) (mM) (mM) (μM)
S Slurry 1 NA NA 130.3 0.0 65.1 162.5 104 3.94
S Slurry 2 5.44 51 0.0 4.6 123.5 NM NM 5.62
M Slurry 1 6.65 54 1.7 0.0 5.3 166.5 45 0.32
M Slurry 2 6.87 56 98.1 0.0 192.9 NM NM 1.09
P Slurry 1 5.89 56 6.6 0.0 118.1 402.0 44 BDL
P Slurry 2 5.82 56 110.1 0.0 205.4 NM NM 0.81
S Source 2 7.73 NM NM NM NM NM NM BDL
Water
P Source 2 7.75 NM NM NM NM NM NM BDL
Water
28 High levels of sulfate and chloride were found in all three systems, with the exception of site M on the first sampling trip (Table 1). Nitrate was undetectable in all three systems, with the exception of site S on the second sampling trip, which was 4.6 mM. Chloride levels decreased from 130.3 mM to 0.0 mM in site S from sampling trip one to two, while chloride at site M increased from 1.7 mM to 98.1 mM and site P increased from 6.6 mM to 110.1 mM. Sulfate increased in systems S and
M, from 65.1 mM to 123.5 mM in site S and 5.3 mM to 192.9 mM in site M. Sulfate decreased in P from 192.9 mM to 118.1 mM. This variability is likely due to differences in water use patterns and changes in chloride and sulfur content in the source coal. The amount of make up water depends on the type of wFGD, unit slurry removal (bleed) rates, and unit loads (higher loads cause more water loss due to evaporation). Coal is sourced from Ohio, West Virginia and Pennsylvania, and most coal burned in these wFGDs is washed first to remove ash, reduce S, and boost energy yield. This is of note since almost all Cl and S found in wFGD systems is introduced through the coal and the concentrations of S and Cl vary within and between coal seams.
Magnesium concentrations for sites S, M, and P were 162.5 mM, 166.5 mM,
402 mM, respectively. Calcium concentration for site S was 104 mM, while for sites
M and P it was 45 mM and 44 mM. The dissolved magnesium and calcium concentrations are lower in site S because it uses limestone, while sites M and P use magnesium-enhanced lime. Site P operates with higher MgOH concentrations
(personal communications). Ferrous iron was below the detection limit in all three wFGD systems. Total mercury was 3.94 and 5.62 μM at site S, 0.32 to 1.09 μM at site
29 M, and below detection level (5-6 μM) to 0.81 μM at site P. Total mercury was below detection level for S source water and P source water.
When viewed as a whole, the wFGD system chemistry reflects that of an extreme environment. All three systems are mildly acidic (pH 5.44-6.87) and operate at high temperatures (51-560C). Although there was variability in chloride and sulfur concentrations within individual sample sites from trip one to trip two, all the wFGD systems had high concentrations on at least one trip, ~90-160 mM.
Magnesium levels were high in all systems, but were over twice as high in site P when compared to sites S and M. The total Hg is low in all three systems, however it is higher in site S than sites M and P, possibly as a result of different water use patterns or source coal.
Microbial Enumerations
Live and dead cells were quantified in all three systems (Table 2). Site S has the lowest overall cell abundances in addition to having fewer live cells than dead cells. Sites M and P have more total cells as well as fewer dead cells than live. Site P had the highest number of live cells and fewer dead cells that site M. (Table 2) The relatively low total cell counts in site S and high ratio of dead:live, may be explained by its acidity (pH 5.44) and elevated mercury content (3.94-5.62 μM) selecting against most microorganisms entering the system from the source water. Site M had higher cell counts and high ratio of live:dead, possibly due to the system pH being closer to that of the source water. The high total cell counts in site P indicate at least some microorganisms in the source water are adapted to the lower pH and the higher operating temperature of the wFGD when compared to the source water.
30 Table 2. wFGD slurry cell counts (cells/mL slurry) by site, using live/dead staining and fluorescent microscopy.
Site Live Cells Dead Cells
(cells/mL slurry) (cells/mL slurry)
S 1.8 x 106 2.7 x 106
M 7.5 x 106 5.4 x 106
P 4.4 x 107 4.9 x 106
31 wFGD Slurry Incubations: Oxygen Consumption Experiment
To assess the aerobic microbial activity with in the systems, I measured O2 consumption rates of microorganisms in the slurries. Total O2 loss over time was measured at temperatures of 20oC, 40oC, 55oC and 70oC with active and deactivated slurry. The microbial communities associated with wFGD systems most likely come from the source water, which varies with the seasons, but does not exceed 25oC
(USGS, 2017). The experimental temperatures were manipulated to simulate source water temperatures (20oC), wFGD temperatures (55oC), and an even more extreme environment (70oC). Samples from the wFGD systems with microbial communities adapted to the temperature and salinity of the wFGD should be most active at 55oC.
Control experiments using Shewanella oneidensis and empty (air only) chambers were conducted at 20oC, 40oC, 55oC, and 70oC to establish activity comparisons for the wFGD systems (Figure 3). The S. oneidensis control experiment established that the respirometer could measure O2 consumption by an active microbial culture and that activity of this organism was dependent on temperature. The control incubations also confirmed that O2 loss was not detected in empty chambers.
32 A B
C D
Figure 3. Oxygen consumption by Shewanella oneidensis, with panels A-D at 20oC, 40oC, 55oC, and 70oC, respectively. Blue = S. oneidensis. Red = empty (air only) chambers.
33 Total O2 loss for site S was very low at all four temperatures, but O2 consumption still increased in active samples with increasing temperature (Figure
4). O2 consumption rates were higher in active site M slurry incubations than
o o o deactivated controls, and O2 consumption rates increased at 40 C, 55 C and 70 C, confirming microbial activity (Figure 5). Site P had the highest O2 consumption rates
o o at all temperatures, but most notably at 40 C and 55 C (Figure 6 J & K). Overall, O2 consumption rates in site S were lowest, but still detectable, and O2 consumption in all sites followed a clear pattern of increased O2 consumption with increased temperature. This indicates active microorganisms that are adapted to the elevated temperatures of wFGD systems.
34 A B
C D
Figure 4. Oxygen consumption by microorganisms associated with site S slurry, with panels A-D at 20oC, 40oC, 55oC, and 70oC, respectively. Blue = active samples, Red = deactivated samples.
35 E F
G H
Figure 5. Oxygen consumption by microorganisms associated with site M slurry, with panels E-H at 20oC, 40oC, 55oC, and 70oC, respectively. Blue = active samples, Red = deactivated samples.
36 I J
K L
Figure 6. Oxygen consumption by microorganisms associated with site P slurry, with panels I-L at 20oC, 40oC, 55oC, and 70oC, respectively. Blue = active samples, Red = deactivated samples.
37 Cell abundances correspond well with the respirometry results. Site S had fewer cells and reflected very low O2 consumption with the respirometer. Site M had
o o more cells and O2 consumption in the 40 C and 55 C temperatures. Site P had the
o highest cell counts overall and the most O2 consumption, most dramatically at 40 C and 55oC. This is especially relevant since the wFGD systems operate at ~55oC, so the most activity (O2 consumption) would be anticipated around that temperature, indicating the microorganisms are specifically adapted to the wFGD environment.
wFGD Slurry Incubations: Mercury Loss Experiment
A total Hg loss experiment was conducted to ascertain whether active slurry samples would retain Hg differently than deactivated slurry samples.
Microorganisms contributing to Hg cycling within the wFGD system would manifest in an increase in total Hg loss in active slurry samples, when compared to inactivated slurry samples. An increase in total Hg loss in active samples would indicate microorganisms are reducing Hg2+ to Hg0, thus volatilizing it.
Active and deactivated slurry samples were amended with 100 μM HgCl2, and then incubated at 57oC for 38 days. Background Hg concentrations in all three systems were 3.94 μM in site S, 0.32 μM in site M, and below detection limit in site P
(Figure 7), while the source water samples were below the detection limit. Hg concentrations decreased in all samples over the 38 days measured. Site S was the only site from which more Hg loss was observed in the active incubations as compared to the deactivated incubations. For site S, days 0-17 Hg loss was similar in active and deactivated samples, however, for days 24-38, active samples lost more
Hg than deactivated samples. Site M Hg loss was similar throughout the 38-day
38 period in active and deactivated samples. Site P also did not demonstrate consistent differences in Hg loss between the active and deactivated samples.
This indicates that, although the cell abundances and aerobic respiration indicate microbial activity in sites M and P, there does not appear to be microbial activity reducing Hg2+ to Hg0 in these systems. An additional consideration is the amended Hg concentration of 100 μM may have been high enough to be toxic to the microorganisms capable of adapting to the chemistry of wFGDs. Some microorganisms from the source water (BDL Hg) may be able to survive the six-fold
Hg increase (6 μM) content of the wFGD, but not the hundred-fold increase used in the experiment. This hypothesis is supported by research conducted with highly Hg resistant marine bacteria that were able to grow in media with Hg concentrations previously established as exceeding survival limits, but could not survive concentrations of 274 μM (Jaysankar et al., 2003).
39
Figure 7. Total Hg concentrations (µmol/L) over a 38-day period. Dark blue = site S active, Red = site S deactivated, Green = site M active, Purple = site M deactivated, Teal = site P active, Orange = site P deactivated.
40 Nucleic Acid-Based Microbial Community Characterization
DNA yields for all three systems were low, however site M contained the most consistently extractable DNA, with the highest yields (Table 3). This is consistent with the cell counts and aerobic respiration, which indicated microbiological activity in site M. The low yields in site P are most likely the result of chemical interactions between the slurry, DNA, and extraction solutions because the cell abundances for site P were quite high, but the DNA yields were consistently low.
Site P has the highest Mg concentrations and Mg is known to bind to DNA due to the opposite charges (Mg is positive, while DNA carries a negative charge). Although this interaction does not necessarily reduce DNA yield, it can render the extracted
DNA inaccessible for downstream applications such as PCR (Bickley et al., 1999).
Washing the slurry samples with TE buffer to remove the Mg marginally improved the DNA yields for site S and M, but not P. This may be due to a different aspect of the slurry chemistry, such as Ca, interfering with Mg removal by TE, or organic PCR inhibitors such as humic, fulminic or tannic acids, heavy metals, or RNases binding the DNA (Schrader et al., 2012). Alternatively, the DNA in site P may be more susceptible to elution during washing or associate less strongly with the pelleted solids, both of which could reduce DNA yields. The lab acquired a Qubit 3.0
Fluorometer (Thermo Scientific Inc., Waltham, MA) between sampling trips, which provided more sensitive measurements. This is reflected in the lower reported DNA yields from the second sampling trip samples.
41 Table 3. DNA concentrations by site and sampling trip.
Site Sampling Trip DNA (ng/μL)
S Slurry 1 2.64
S Slurry 2 0.45
M Slurry 1 1.90
M Slurry 2 0.21
P Slurry 1 1.13
P Slurry 2 0.18
S Source Water 2 5.22 X 104
P Source Water 2 3.28 X 104
42 In total, 743,963 sequences were recovered from the wFGD and source water samples. Of those, 144,145 were recovered from wFGD systems on the first sampling trip and 329,893 sequences on the second trip. An additional 269,925 sequences were recovered from the source water samples collected on the second sampling trip. Overall, more sequences were recovered on the second sampling trip than the first, along with a corresponding increase in the number of unique OTUs identified, in each wFGD system (Table 4).
Microbial community diversity, as indicated by Shannon richness estimates, was higher in the source samples than the wFGD samples (Table 4). This indicates the source water (river) has a more diverse microbial community than the wFGD slurry. Within the slurry samples, site M had the lowest Shannon values suggesting that the associated microbial community is least diverse. This could correspond to a microbial community that is more limited, but better adapted to the temperature, pH, and salinity within the wFGD, when coupled with the O2 consumption and cell counts indicating microbial activity.
43 Table 4. Estimates of microbial abundances and diversity of wFGD systems.
Site Sampling No. of No. of unique Shannon Index
Trip Sequences OTUs
S Slurry 1 57,186 706 5.17
S Slurry 2 102,895 2,991 6.02
M Slurry 1 51,989 521 4.17
M Slurry 2 140,147 2,728 4.49
P Slurry 1 34,970 609 5.34
P Slurry 2 86,851 1,665 4.54
S Source Water 2 135,615 4,123 6.80
P Source Water 2 134,310 4,092 6.08
44 Rarefaction analysis was conducted to assess sampling depth by graphing the average number of observed OTUs against the number of sequences per sample
(Figure 8). The two source water microbial communities were the least thoroughly sampled, as demonstrated by the height and slope of the lines in the graph. This agrees with the higher number of sequences recovered and higher Shannon values from the source samples, all indicating a more diverse microbial community. The wFGD sequence data from the first sampling trip contained fewer total sequence reads on average for all three sites than the second sampling trip, explaining why they appear more deeply sampled. The rarefaction curves suggest sampling of the wFGD systems is approaching saturation.
45
Figure 8. Graph of rarefaction analysis for wFGD systems from both sampling trips and source water. Dark blue = S1, red = M1, green = P1, purple = S2, teal = M2, orange = P2, light blue = SW, and pink = PW.
46 At the phylum-level, Proteobacteria and Actinobacteria were abundant in the source water samples (Figure 9 A). 10.3% of S source water and 7.7% of P source water of recovered sequences were Betaproteobacteria. The source water contained high relative abundances of Alphaproteobacteria, 31.8% of recovered sequences for
S source water and 20.7% for P source water. Firmicutes comprised less than 1% of recovered sequences in both the source water samples. Sites S and P source water recovered sequences contained 12.3% and 8.6% Cyanobacteria, respectively. Site S and P source waters were similar in that they both contained abundant
Alphaproteobacteria, Betaproteobacteria, and Cyanobacteria. However, S source water also contained abundant Acidobacteria (10.5%).
Fewer total taxa were observed at the phylum-level in the wFGD systems when compared to the source water (Figure 9). With the exception of site P on the first sampling trip, which had nearly equal relative abundances of Cyanobacteria and Proteobacteria, Proteobacteria OTUs were most abundant all wFGD samples. Of the wFGD samples, Betaproteobacteria consistently composed nearly half of the recovered sequences on both sampling trips, and was similar to the recovered sequences found in the source water samples (Figure 9 A). Relative abundances of
Alphaproteobacteria in all sites on the first sampling trip were much lower than those found the following year in the wFGDs and in the source water samples
(Figure 9 A). The relative abundances of Firmicutes in recovered sequences also dramatically decreased between the first and second sampling trips, and were very low (under 1%) in the source water samples (Figure 9 A). Gammaproteobacteria
OTU abundances decreased in site S and M between sampling trips and increased
47 slightly in site P (Figure 9 A). The dramatic shifts in phylum-level relative abundances were higher than expected given the tightly controlled operation of wFGD systems for optimal SOx removal.
All three wFGD associated microbial communities had higher relative abundances of Cyanobacteria-affiliated phylotypes on the first sampling trip (Figure
9 A), causing the wFGD sites to appear more similar than they are. However this is misleading, because the organisms found in the wFGD samples and taxonomically assigned as Cyanobacteria, were actually chloroplasts from photosynthetic eukaryotic organisms. Since these organisms are unlikely to be active in a lightless wFGD tank, and can obscure potentially active microorganisms, I removed them.
After removing the chloroplast-attributable sequences from all wFGD samples
(Figure 9 B), the general trend of the systems being more similar to each other within sampling trips than to themselves across sampling trips, remains.
Alphaproteobacteria increased in relative abundance on the second sampling trip.
Since the source water and the lime or limestone are the primary inoculum for the wFGD systems, dramatic shifts in source water composition could cause apparently large, but ultimately insignificant, shifts in wFGD system compositions as reflected by 16S rRNA gene sequencing data, which picks up all DNA, not just the
DNA found in live and active cells. Therefore, the wFGD could appear to have large shifts in composition that are not realistic reflections of shifts in the active microorganisms, and would explain the shift seen in the wFGD systems between the first and second sampling trips. The fact that all wFGD samples are more similar to each other based on sampling trip, than they are to themselves across sampling
48 trips, and the fact that the source water samples and all the wFGD samples from the second trip are much more similar, supports this conclusion. The higher relative abundances of Betaproteobacteria and Firmicutes in all wFGD systems as compared with the source water samples indicates the wFGD environment is and enriching microorganisms within those lineages. Both Betaproteobacteria and Firmicutes have known thermophilic species (Hedlund et al., 2012; Miyake et al., 2007).
49 A
B
Figure 9. Phylum-level (class-level for Proteobacteria) OTU relative abundances for bacterial 16S rRNA genes. Frame A includes chloroplast-attributable sequences (Cyanobacteria) for all samples. Panel B includes chloroplast-attributable sequences (Cyanobacteria) in source water samples only.
50 Microbial communities from all wFGD systems for both sampling trips and the source water samples were compared visually using weighted and unweighted
UniFrac PCoA plots, calculated first with Cyanobacteria, and then without. These
Cyanobacteria are actually chloroplast-attributable sequences and were removed since their presence in the wFGD system in unlikely to translate into activity given the lightless interior. For this analysis, samples are labeled with the first letter of the site and 1 or 2, designating sampling trip (i.e. site M, trip one = M1). For all PCoA plots, SW and PW clustered, except for the weighted metric PCoA with chloroplast- attributable sequences removed (Figures 10 and 11). S1, M1, and to a lesser extent
P1, cluster tightly in both weighted (Figure 10 A-C) and unweighted PCoA plots with chloroplast-attributable sequences (Figure 10 D-F), and the unweighted PCoA plots without chloroplast-attributable sequences (Figure 11 J-L). S2 and M2 also cluster in all except the PC1 vs. PC3 and PC2 vs. PC3 weighted PCoA plots without chloroplast- attributable sequences (Figure 11 H & I). The consensus of all the unweighted PCoA plots, and the weighted with chloroplast-attributable sequences (Figure 10 A-E &
Figure 11 F-H), is a distinct clustering pattern of S1, M1, and usually P1. S2 and M2 are also closely associated, as are SW and PW with each other. P2 does not associate with any other samples. In contrast, S1 and S2, M1 and M2, and P1 and P2 cluster in the weighted PCoA plots without chloroplast-attributable sequences for PC1 vs. PC2
(Figure 11 G). P1, P2, and S2; and M1, M2, and S1 cluster in PC1 vs. PC3 (Figure 11
H). P1 and P2, and M1, S1, and PW cluster in PC2 vs. PC3 (Figure 11 I).
Weighted metrics take into account the presence or absence as well as the abundance of observed OTUs, while unweighted metrics only take into account the
51 presence or absence of an OTU. This explains the observed patterns of clustering seen in the PCoA plots, since when present, the chloroplast-attributable sequences are so abundant that they obscure differences between the samples, rendering them similar with regard to total taxa present and their relative abundances that they group according to sampling time, regardless of metric. This suggests the operating conditions, especially water use patterns, and not the extreme environment of the individual wFGD unit, is most important in shaping the microbial communities they host. However, when relative abundance (weighted metrics) are used and chloroplast-attributable sequences are removed, samples group by sample site rather than sample time. This suggests that wFGD unit chemistry likely shapes the microbial communities associated with them.
52 A D
B E
C F
Figure 10. PCoA analysis of wFGD associated communities and source water samples. A-C use weighted metrics. D-F use unweighted metrics. A = PC1 vs. PC2, B = PC1 vs. PC3, C = PC2 vs. PC3, D = PC1 vs. PC2, E = PC1 vs. PC3, F = PC2 vs. PC3
53 G J
H K
I L
Figure 11. PCoA analysis of wFGD associated communities and source water samples with chloroplast-attributable sequences removed. G-I use weighted metrics. J-L use unweighted metrics. G = PC1 vs. PC2, H = PC1 vs. PC3, I = PC2 vs. PC3, J = PC1 vs. PC2, K = PC1 vs. PC3, L = PC2 vs. PC3.
54 This apparent shift in system taxonomic composition between sampling trips across all three sites at the phylum-level is not reflective of a dramatic overall decrease in taxa present within the wFGD units at the genera-level (Figure 12). All genera present above 5% of the recovered sequences are shown, along with those genera present at less than 5%, grouped into a single category. The top orange band in Figure 12 is a grouping of multiple individual genera that composed less than 5% of recovered sequences from a sample, almost all of which were actually under 1% of recovered sequences. Since the individual genera in this grouping have such low relative abundance, it is likely that their activity is minimal in the context of the wFGD system.
The two source water samples are almost identical even though the sites are over 100 miles apart. When looking at genera present above 5% of total sequences, the source water samples shared the same four genera, in similar relative abundances. S source water and P source water contained, respectively, 9.5% and
10.2% CL500-29 marine group (an Acidimicrobiaceae), 21.0% and 20.0% hgcl clade of Sporichthyaceae, 9.4% and 6.9% an uncultured chloroplast, and 22.0% and 33.0% uncultured bacterium in the SAR11 clade of Alphaproteobacteria.
When considering all genera present above 5% in any one system (Figure
12), sites S and M genera decreased from 5 on the first trip to 3 on the second. Site P genera increased from 5 on the first trip to 7 on the second. Of the genera present, only one was shared by both the source water samples and a sample from the wFGD systems. This was site S that shared 5.3% of the hgcl clade from the Sporichthyaceae family. Although sequences attributable to chloroplasts (taxonomically grouped
55 under the Cyanobacteria phylum) were present in all samples, the genera and relative abundances varied. All three wFGDs shared an uncultured chloroplast on the first sampling, and site P contained an additional chloroplast, Phaseolus acutifolius. None of the wFGD samples from the second trip had chloroplasts above
5%, while the source water samples contained a different chloroplast taxonomically assigned to an uncultured bacterium.
Of the wFGD systems, only one genus was shared by all three sites on both sampling trips, when comparing the relative abundances present above 5%: an unidentified chloroplasts, which composed 8.1%, 10.0%, and 21.8%, respectively of sites S, M and P (Figure 12). Hydrogenophilus and Thiobacillus, both from the thermophilic Hydrogenophilaceae family, were found in the wFGDs (Figure 12 mint and bright blue bands). Site M had 36.8% Hydrogenophilus on the first sampling trip and 38.9% on the second sampling trip. Site S had 26.3% Hydrogenophilus on the first trip and 19.0% Thiobacillus from the second sampling trip. The S source water sample contained 0.2% Hydrogenophilus and 0.3% Thiobacillus, while P source water had 0.3% Hydrogenophilus and 0.7% Thiobacillus. Hydrogenophilus in particular is known to be a H2 oxidizing thermophile, while Thiobacillus is a S oxidizing thermophile.
56
5 7
Figure 12. Genera-level of all OTUs from bacterial 16S rRNA genes found above 5% in wFGD and source water samples. All genera under 5% grouped and represented by top orange band.
57 The 16S rRNA gene sequencing data indicates that the wFGD systems at sites
S and M have retained present, but rare, genera from the source water and provided them with a suitable environment to become over a third of the microbial community present. Additionally, and perhaps of more interest, the genera enriched in the wFGD systems and found at such a high relative abundances are likely the source of the activity found in the other experiments. Specifically the cell counts and increased O2 consumption at high temperatures in the active slurry samples indicate the wFGD systems enrich thermophilic lineages.
16S rRNA gene transcript characterization
Total RNA from wFGD slurry samples collected on the second sampling trip was sequenced using the 16S rRNA gene sequences to approximate microbial activity in wFGD systems. Over 90% of total RNA is ribosomal RNA (rRNA), and as such, can be used as a proxy for the total ribosomal content of a cell, and by extension, the activity potential for that cell (Blazewicz et al., 2013). The total RNA reads per sample can be compared at the taxa-level to assess potential for activity by taxa within and across samples. The returned sequencing data was assembled de novo using Rockhopper and sequences with high numbers of transcript reads were searched using the NCBI database BLASTN tool to identify microorganisms (Altschul et al., 1997).
A total of 266 transcripts were assembled from sequence reads found in all three samples (Table 5). The transcripts had an average length of 986 base pairs and a median length of 374 base pairs. Of the total assembled transcripts, site S had
16, site M had 166, and site P had 89. The average number of reads per assembled
58 transcript, by sample site, was 5.5E+16 for site S, 236 for site M, and 1086 for site P.
Site S had the fewest total assembled transcripts, as well as the most reads per transcript. The higher total reads, more perfectly aligned reads, and higher total number of assembled transcripts in site M indicate that this site has more ribosomes and thus likely more, more active taxa present.
59 Table 5. Total and perfectly aligned reads produced by de novo assembly of total RNA from wFGD sample sites.
Site Total Reads Perfectly Aligned Total Assembled Average
reads Transcripts Reads/
Transcript
S 3,931,531 14,921 (0%) 16 5.5E+16
M 4,288,805 48,149 (1%) 166 236
P 3,562407 28,764 (1%) 89 1086
60 However, this number of total assembled transcripts is not entirely accurate since many of the assembled sequence reads were matched to eukaryotic sequences using the NCBI BLASTN tool and are therefore of little interest in ascertaining microbial community activity. Once sequence reads that mapped to eukaryotes were removed, site S had 7, site M had 5, and site P had 67 non-eukaryotic assembled sequences. I removed sequences that only matched to bacterial cloning vectors, phages and viruses, and then reevaluated the remaining sequences
(Appendix 1). Although not specifically known to be thermophilic or halophilic, several species from genera with known Hg metabolizing capabilities were found.
These included sequence matches to Pseudomonas and Desulfitobacterium from all three sites, as well as Actinobacter and Geobacter from site P.
Of those sequences, few sequences matched to known halophilic or thermophilic species (Appendix 1). However, site S had a sequence match to
Marinobacter salinus, a halophilic, sulfur oxidizing, aromatic carbon degrader isolated from marine sediments. Three sequences were observed in site M with matches to thermophilic species: Thermonospora curvata DSM 43183, an aerobic thermophile with optimal temperature of 65oC and pH of 6; Desulfotomaculum kuznetsovii 17 DSM 6115, an obligate anaerobic mesophilic sulfur-reducer with an optimal temperature of 60oC; and Thermus thermophilus HB8, an aerobic, halotolerant thermophile with and optimal temperature of 85oC, isolated from a freshwater hot spring. Lastly site P returned sequence matches to Cupriavidus necator, a hydrogenotroph.
61 Although site M had the fewest total non-eukaryotic assembled transcripts, it had the most matches to thermophilic species. This presents a strong case for active microorganisms in site M when viewed along side the previous cell count, respirometry, and 16S rRNA gene analysis.
62 CHAPTER IV
DISCUSSION
This research determined the chemical characteristics of wFGD slurries, as well as the composition and activity of the associated microbial communities, and the potential for microbial Hg reduction within wFGDs. Hg is toxic to human health in all its forms, and is known to damage the central and peripheral nervous systems in adults and impair natal development (Ozuah 2000). MeHg is the most prevalent form of organic Hg in nature, and is readily bioaccumulated in food webs (Gochfeld
2003). This leads to increased human exposure through the concentration of MeHg with increased trophic levels. In the US, coal fired power plants are the single largest anthropogenic contributor of environmental Hg, which is more likely to be methylated once deposited in bodies of water and anoxic soils (USEPA, 2016).
As air pollution control regulations become increasingly stringent, utilizing the Hg reducing activity of microbial communities is an attractive alternative to developing new chemical or mechanical Hg treatments. Based on what is currently known about Hg metabolism, microbial activity could affect Hg entrainment in wFGD systems in one of two ways: oxidation of Hg0 to Hg2+ or methylation of Hg to
MeHg, thus increasing overall Hg retention, or reduction of Hg2+ to Hg0, thereby increasing overall atmospheric Hg emissions (Boyd & Barkay, 2012; Hsu-Kim et al.,
63 2013). However, little is known about what microorganism are even present in wFGD systems, much less their activity (Brown et al., 2012). Once the microbial activity of wFGDs is established, their role in Hg speciation and potential to boost Hg retention can be addressed. Therefore, the first step to controlling microbially mediated Hg speciation in wFGD systems is characterizing the microbial community present and identifying the active contributors.
In order to address the question of Hg metabolism within wFGD systems, we first determined whether microbial communities are active in the extreme wFGD environment. This research provides evidence supporting the presence and activity of microbial communities within wFGD slurry. The activity of these microorganisms requires some adaption from the source environment in response to the wFGD environment. Microbial communities introduced into the extreme environment of the wFGD system from physiochemically moderate river systems changed in diversity and relative abundances of represented taxa. The dramatic enrichment for thermophilic species within the wFGDs when compared to the source water indicated adaption of microbial communities from the source water.
Microbial Adaptability to Extreme Environments
Although research specifically studying the adaption of previously mesophilic microbial communities to extremely hot and saline environments is scarce, it has been established in previous research that microorganisms contain a wide array of metabolic pathways that enable them to adapt to changes in their immediate environment or available energy sources by up- or down- regulating gene expression or switching terminal electron acceptors (Kogut 1980; Konhauser
64 2007). Furthermore, there is some relevant work focused on improving bioreactor performance and soil ecosystem function through the associated microbial communities’ response to anticipated climate change. The bioreactor study found that the community structure and function of microbial communities changed in response to shifts in nutrient loads (Ahn et al., 2006). In the case of soil communities, increased temperatures correlated with increased metabolic activity up to a point, after which the microorganisms were unable to maintain cellular function and died en mass (Birgander et al., 2013; Liu et al., 2015; Wallenstein &
Hall, 2012). Lastly one study introduced freshwater microbial communities into saline environments and found that the overall structure of the microbial communities shifted such that once rare or dormant halophilic taxa became the most abundant taxa (Zhang et al., 2014).
Additional research conducted on microbial communities in geothermal systems and hot springs is relevant to my findings based on similarly extreme physiochemistry (Burton & Norris, 2000; Neveu et al., 2016). Hot springs, in particular, have diverse microbial communities that thrive at elevated temperatures, increased acidity, and high metal and salt concentrations. Some
Yellowstone hot springs have chemistries vary similar to wFGD systems and support microbial communities successfully utilizing a wide range of metabolic strategies (Shock et al. 2010).
The chemistry of my wFGD systems is similar to that found in Brown et al.
(2012) in general physiochemical trends and microbial community composition.
The wFGD slurry from this research was slightly more acidic, with overlapping
65 - 2- 2+ 2+ operating temperatures and similarly variable Cl , SO4 , Ca , and Mg concentrations. The dissolved ion concentrations in my study and in comparison to those reported by Brown et al. (2014) are variable, but generally, were all higher than the source water, and combined with the pH, constitute a corrosive environment. The wFGD physiochemistry as a whole reflects an extreme, but variable environment.
Microbial source water and wFGD slurry relative abundances of OTUs, as determined through 16S rRNA gene sequencing, are quite different from each other, which is consistent with previous research (Brown et al., 2012). However, the taxa and relative abundances differed, both within my project and between my research and Brown et al. (2012). This can be partially explained by addressing sampling depth, but may be a reflection of 16S rRNA gene sequence databases (Greengenes vs. SILVA) used in the two studies. The Shannon Index values were lower in both studies when comparing source water to wFGD slurry samples. This indicates the wFGD systems are consistently less diverse than the source water, further supporting the hypothesis that wFGD microbial communities are shaped by the extreme environment.
Additionally, PCoA plots of all three systems were constructed using the weighted and unweighted UniFrac metric, with and without chloroplast-attributable sequences. Weighted metrics in UniFrac are calculated with abundance in addition to simple presence/absence of taxa, while unweighted metrics account only for presence/absence of taxa. The analyses were skewed by chloroplast-attributable
16S gene sequences, since photosynthetic organisms are unlikely to be active in
66 lightless environments, and as such, could be excluded from analyses. The resulting
PCoA plots support this hypothesis. When chloroplast-attributable sequences are present, the wFGD systems group according to sampling trip, regardless of metric.
When chloroplast-attributable sequences are removed, the unweighted plots associate strongly with sampling trip, while the weighted plots cluster by site. Once the likely inactive chloroplasts are excluded, the samples clustered by site with a weighted metric, much like the PCoA plots constructed by Brown et al., which indicates the physiochemisty of the wFGD enrich for microbial communities distinct from their source water (2012).
The relative abundance of phylum-level taxa showed similar patterns, with
Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and
Firmicutes comprising the majority of OTUs in wFGD samples from both studies. The
Firmicutes-associated OTUs had higher relative abundances in the wFGD samples when compared to the source water in both studies. However, in my research, the
Betaproteobacteria were also elevated in the wFGD slurry with regard to the source water. The high relative abundance of chloroplast-attributable sequences in the wFGD and source water samples from my second sampling trip are not reflective of the previous study, where Cyanobacteria (which were not identified as chloroplast- attributable sequences by Brown et al.) were only recovered from one wFGD slurry and were extremely low in the source water. This was attributed to the recent restarting of the wFGD unit in question, however, that is an unsatisfactory explanation in light of my findings. It is much more likely the chloroplast- attributable sequences vary with source water conditions and/or the seasons and as
67 a direct result, the wFGD systems shift. Although it is important to note this shift is unlikely to indicate a change in the active microbial community, since the
Cyanobacteria are chloroplast-attributable sequences and thus are unlikely to be active in the dark wFGD.
Genera-level taxa identified in this project differ from the Brown et al. (2012) study, especially with regard to thermophilic linages. Only one genus was found in both the previous study and mine: Thiobacillus. However, Brown et al. (2012) only observed it in the source water, while I found it in the two out of three wFGDs and both source water samples. Of particular interest are two genera of
Betaproteobacteria with high relative abundance in both site S and site M on both sampling trips. Hydrogenophilus composed 26.3% (trip one) and 2.6% (trip two) in site S, and 38.9% (trip one) and 36.8% (trip two) in site M. Thiobacillus composted
7.1% (trip one) and 19.0% (trip two) in site S, and less that 1% both trips for site M.
The source water samples were composed of less than 1% of both genera, both trips.
These genera belong to the family Hydrogenophilaceae and are Gram negative, rod shaped, thermophilic, and chemolithotrophic (Orlygsson and
Kristjansson 2014). Thiobacillus sp. have been isolated from freshwater, marine and estuarine sediments, aquifers, sulfur springs, and soil, is active up to 55oC and the genus includes species capable of both aerobic and anaerobic respiration when provided with S-compounds (Orlygsson and Kristjansson 2014). Hydrogenophilus was originally isolated in hot springs, is aerobic, and can oxidize hydrogen
(Orlygsson and Kristjansson 2014). The likelihood that there are species of this
68 genera active in the wFGD slurry is reinforced by the successful cultivation of hydrogen oxidizing, sulfate reducing microorganisms in hydrogen enriched media inoculated with site M sample slurry (not shown).
wFGDs are dynamic systems, which is reflected in changes in chemistry, most notably dissolved ions, and taxa relative abundances. These changes in dissolved ions are most likely the result of changes in source coal and possibly limestone/magnesium-enhanced lime. The microbial community composition may also shift in response to changes in system chemistry, in the microorganisms arriving from the river, or in water use patterns (Zhang et al., 2014; personal communication with Mark Golightly). However, the Hydrogenophilus and
Thiobacillus abundances were high in both sampling and were much higher than the abundances found in the source water. It seems likely that these genera are a major active component of the slurry communities and are oxidizing H2, and oxidizing or reducing S.
Implications of Findings
In the US, atmospheric Hg emissions from fossil fuel combustion are the single largest anthropogenic contributor to atmospheric Hg and thus increase total
MeHg production by microorganisms (Driscoll et al., 2013; USEPA, 2016). The industrial revolution and subsequent increase in demand for fossil fuels, especially coal, brought Hg that had been trapped in the lithosphere to the surface. The high combustion temperatures of coal fired power generation results in the reduction of bound Hg2+ to highly volatile Hg0, which then escapes to the atmosphere, where a portion of it is deposited onto the earth’s surface (Dίaz-Somoano et al., 2007).
69 Eventually some of the Hg makes its way into anoxic saturated sediments and lower portions of water columns where microorganisms with Hg reducing or methylation capabilities reside (Hsu-Kim et al. 2013). Coal combustion has led to a net increase in Hg participating in the Hg cycle and is, therefore, a major contributor to the rise in
MeHg (Driscoll et al. 2013).
Although significant advances have been made to control most forms of air pollution from coal combustion (e.g. SOx, NOx, and particulate matter), there are no
0 technologies specific to Hg removal. Hg is primarily found in wFGDs as Hgp, Hg , and
Hg2+, with the speciation driven largely by slurry chemistry. Hg2+ reduction to volatile Hg0 increases the amount of Hg released into the atmosphere. As governmental regulations continue to decrease the maximum Hg emissions allowable from coal fired power plants, the available mechanical and chemical air pollution control (APC) technology increasingly struggles to meet ever more stringent Hg emission regulations. This makes the possibility of microbial means of reducing Hg emissions increasing appealing to power companies and others concerned with Hg pollution.
wFGD Microbial Mercury Metabolism
Overall wFGD system Hg retention could be enhanced by limiting the presence and activity of Hg reducing microorganisms, and/or by increasing the presence and activity of Hg oxidizing microorganisms within the wFGD unit
(Chapter 1)(Zhang et al., 2016). Microorganisms reducing Hg2+ to Hg0 in the wFGD system will contribute to higher total Hg0 emissions from coal fired power plants.
The net result of which will be increased Hg in the atmosphere, and eventually
70 increased environmental MeHg. Alternately, microbial Hg oxidation of Hg0 to Hg2+ will boost Hg retention in wFGD systems, lowering the amount of Hg emitted from coal combustion.
Lastly, the Hg2+ captured in the wFGD system, could be methylated and/or demethylated by microorganisms in the slurry. Methylation would be beneficial for capture, but could lead to downstream treatment problems. Increased Hg levels have been documented in wallboard produced from synthetic gypsum, and MeHg would be even more difficult to remove from slurry wastes or byproducts (Kairies et al., 2006). While Hg demethylation could transform MeHg to Hg0, once again increasing Hg emissions and net atmospheric Hg0, and ultimately increasing environmental MeHg levels.
The genes responsible for Hg reduction, oxidation, and methylation in microorganisms have been identified, and Hg metabolizing organisms have been found in many systems. Sulfate-reducing (SRB) and iron-reducing-bacteria (FeRB) were among the first Hg methylating bacteria identified, but many other species have been found by targeting the genes specific to Hg methylation, specifically hgcA and hgcB (Parks et al., 2013). Many additional syntrophic Deltaproteobacteria,
Firmicutes, and methanogens have been identified using the hgcA/B genes, and it is likely that Hg methylation is a function of many microorganisms in anaerobic environments (Podar et al., 2015). Since I found SRB and Firmicutes in both my 16S rRNA sequencing and RNA sequencing data, it is possible these organisms are capable of carrying out Hg methylation in the wFGD environment.
71 The genes responsible for Hg reduction and demethylation, found in the mer operon, are present in 1-10% of cultured heterotrophic, aerobic microorganisms, as well as in Proteobacteria, Firmicutes, Actinobacteria, Crenarchaeota, Aquificae, and
Thermus/Deinococcus (Boyd and Barkay, 2012). The wFGD systems evaluated in my research contained several lineages linked to the mer operon, including Firmicutes and Proteobacteria (Pseudomonas), based on 16S rRNA gene sequencing and RNA sequencing data. Given the number of microorganisms currently known to metabolize Hg, and the large number of microorganisms still unknown, it is likely that they are already present in, or could be successfully introduced into wFGD systems (Boyd and Barkay 2012). These wFGD associated microbial communities have the potential for manipulation since they are active and appear stable. Thus Hg oxidizing species could be introduced or enhanced in wFGDs to increase Hg retention, and Hg reducing species should be inhibited to decrease Hg emissions through the exhaust stack.
Future Directions
I found moderate numbers of live cells, aerobic activity, and 16S rRNA gene sequencing evidence of thermophilic lineages of bacteria in the wFGD slurries, all of which strongly indicate microbial activity in wFGD systems. Additionally, a H2 oxidizing and sulfate reducing bacterial enrichment was successfully developed at
500C from site M slurry. Although this research presents a strong case for the presence of active microbial communities within wFGDs, the question of what specific microbial metabolisms are active and how those metabolisms are influencing wFGD performance remain.
72 Additionally, the unresolved question of whether microorganisms can be manipulated to reduce wFGD Hg emissions is unresolved, and broader questions regarding the role of microorganism metabolisms causing or increasing the wFGD tank corrosion, contributing to SOx or CO2 emissions, or silica formation should be addressed. The first step towards addressing the above questions, is continuing enrichment cultures and sequencing efforts to better understand what organisms are active and present in wFGDs and clarify active metabolic pathways. Further efforts are needed to culture and isolate additional microorganisms from wFGD slurry for individual genus and species level identification.
73 CHATPER V
CONCLUSION
MeHg is a major human health issue, and is of increasing concern due to the rising amount of Hg participating in global Hg cycles, primary through coal combustion for electricity generation. Because the flue gas from coal combustion furnaces all pass through exhaust treatment systems, microorganisms in wFGD units are ideally situated to enhance Hg capture. The microbial communities found in wFGD slurry can potentially be manipulated to reduce net Hg emissions from coal fired power plants by enrich for, or select against, specific genera. This research is a convincing first step in establishing the chemistry of wFGD systems in addition to the presence of active microorganisms systems and the potential for microbial Hg metabolism.
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81 APPENDIX
Non-eukaryotic assembled transcripts by wFGD site
Si Seque Len Expres Top Sequence Sequence Sequence Sequence Sequence te nce gth sion Sequenc ID Match ID Match ID e Match S 1 Pseudo Aerobic, Marinobac Halophili monas mesophil ter salinus c, S- putida e, Mn- oxidizing, GB-1 oxidizer aromatic hydrocar bon 1.4411 degradin 64 5E+17 g S 2 Pseudo Aerobic, Marinobac Halophili monas mesophil ter salinus c, S- putida e, Mn- oxidizing, GB-1 oxidizer aromatic hydrocar bon 1.3974 degradin 66 8E+17 g S 3 Maribac 5.4901 ter sp. 168 E+16 T28 S 4 Delftia Aerobic, Cs1-4 pH 5.5- 7.5, 30oC, 1.8192 heterotro 507 1E+16 ph S 5 Delftia Aerobic, Cs1-4 pH 5.5- 7.5, 30oC, 1.8120 heterotro 509 6E+16 ph S 6 Desulfit Anaerobi obacteri c, um mesophil 538 1.7124 hafniens e 6 7E+15 e M 1 Pseudo Thermono Aerobic, monas spora pH 6, sp. CCOS curvata Thermop 191 DSM hile 65oC 87 1113 43183
82 Non-eukaryotic assembled transcripts by wFGD site continued
M 2 Desulfit Anaerobi obacteri c, um mesophil 538 hafniens e 6 677 e M 3 Tessara Desulfoto Obligate Thermus Aerobic, coccus maculum anaerobe, thermop Halotoler flavesce kuznetsovi mesophil hilus HB8 ant, ns DSM i 17 DSM e 60oC, Thermop o 18582 6115 H2S gas hile 85 C, release, freshwat 57 206 S-reducer er M 5 Burkhol Stenotrop Aerobic, deria homonas mesophil pseudo maltophili e multivo a R551-3 53 136 rans P 1 Desulfit Anaerobi obacteri c, um mesophil 538 hafniens e 6 6008 e P 2 Desulfit Anaerobi obacteri c, um mesophil 502 hafniens e 8 1742 e P 3 Desulfit Anaerobi obacteri c, um mesophil 505 hafniens e 8 1645 e P 4 269 Escheric 7 1589 hia coli P 5 266 Escheric 8 1571 hia coli P 6 Methylo Aerobic, bacteriu mesophil m e 425 extorqu 8 1373 ens AM1 P 7 Xantho Aerobic, Cupriavidu hydrogen monas mesophil s necator otroph campest e ris pv. 400 campest 8 1340 ris
83 Non-eukaryotic assembled transcripts by wFGD site continued
P 8 Xantho Aerobic, Cupriavidu hydrogen monas mesophil s necator otroph campest e ris pv. 519 campest 9 1328 ris P 9 Acinetobac Aerobic, ter mesophili Pseudo calcoacetic c, 169 monas us PHEA-2 chemohet 3 1137 sp. JY-Q erotroph P 10 Burkhol 179 deria 3 1137 lata P 11 Weissell a 200 jogaejeo 9 1078 tgali P 12 Acinetobac Aerobic, ter mesophili Pseudo calcoacetic c, 168 monas us PHEA-2 chemohet 8 1045 sp. JY-Q erotroph P 13 Weissell a 118 jogaejeo 5 1018 tgali P 14 Klebsiell a variicol 539 957 a P 15 Roseom onas 367 951 gilardii P 16 Anaerobi c, Corynebac mesophili Colwelli teriuum c, a sp. ammoniag chemoor PAMC enes DSM ganotrop 338 915 21821 20306 h P 17 Geobacter Anaerobi Anaerobi sulfurredu c, c, cens PCA mesophili mesophili c, c, chemolit chemolit hotroph, Geobacte hotroph Synecho toxic r cystis metal and sulfurred sp. PCC S- ucens 418 897 6714 reducing KN400
84 Non-eukaryotic assembled transcripts by wFGD site continued
P 18 Bordetel Corynebac Aerobic, la terium mesophili pseudoh imitans c, inzii chemoor ganotrop 613 894 h P 19 Olsenell a sp. oral taxon 425 883 807 P 20 Bartone lla schoenb uchensis 459 881 R1
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