







































Aresty Rutgers Undergraduate Research Journal, vol. 1, issue 1, Spring 2020 

 

 
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. 

Characterizing and Evaluating the Diverse 

Microbial Communities and Their Mercury 

Resistance Potential from Hot Spring Sites 

Representing Gradients in Temperature and pH 

in Yellowstone National Park  
 

Yelizaveta Rassadkina, Spencer Roth, and Tamar Barkay 

Department of Biochemistry and Microbiology, Rutgers University 

 

Abstract 

 

Yellowstone National Park is home to many different hot springs, lakes, geysers, pools, and 

basins that range in pH, chemical composition, and temperature. These different environmental 

variations provide a broad range of conditions that select and grow diverse communities of 

microorganisms. In this study, we collected samples from geochemically diverse lakes and 

springs to characterize the microbial communities present through 16S rRNA metagenomic 

analysis. This information was then used to observe how various microorganisms survive in 

high mercury environments. The results show the presence of microorganisms that have been 

studied in previous literature. The results also depict gradients of microorganisms including 

thermophilic bacteria and archaea that exist in these extreme environments. In addition, beta 

diversity analyses of the sequence data showed site clustering based primarily on temperature 

instead of pH or sample site, suggesting that while pH, temperature, and sample site were all 

shown to be significant, temperature is the strongest factor driving microorganism community 

development. While it is important to characterize the microorganism community present, it is 

also important to understand how this community functions as a result of its selection. Along 

with looking at community composition, genomic material was tested to see if it contained 

mercury methylating (hgcA) or mercury reducing (merA) genes. Out of 22 samples, three of 

them were observed to have merA genes, while no samples had hgcA genes. These results 

indicate that microorganisms in Mustard and Nymph Springs may use mercury reduction. 

Understanding how microorganisms survive in environments with high concentrations of toxic 

pollutants is crucial because it can be used as a model to better understand mechanisms of 

resistance and the biogeochemical cycle, as well as for bioremediation and other solutions to 

anthropogenic problems.  

 

Key terms 

Chemolithotrophs, mercury methylation, mercury reduction, thermophiles. 

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https://creativecommons.org/licenses/by-nc-sa/4.0/


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Introduction 

 

Yellowstone National Park is well known for its unique aquatic geothermal habitats, which 

exhibit a wide array of extreme temperatures and pH levels. Extreme conditions select for 

lithotrophic extremophiles, organisms that obtain reducing equivalents from inorganic 

substrates (Cuhel et al., 2002). Chemolithotrophs are a specific group of lithotrophic organisms 

that use inorganic substrates for ATP production, as well as inorganic material for carbon 

compound biosynthesis such as CO2 fixation (Cuhel et al., 2002). These groups of organisms 

belong to the archaeal and bacterial domains and are dominant in the extreme environments 

common in Yellowstone National Park (Cuhel et al., 2002).  

 

Yellowstone’s geothermal activity leads to the presence of hydrothermal springs, which emit 

compounds such as sulfide, elemental sulfur, arsenic, and iron (Shock et al., 2010). These 

compounds can then be used as a source of energy for local microorganisms. Since most of the 

microorganisms in hot springs are chemolithotrophic thermophiles, they require specific 

nutrients and extreme conditions that are not easily replicated in a laboratory setting. 

Therefore, the ecology of microbial communities within Yellowstone is not as well-

characterized as communities from many mesophilic environments (Reysenbach et al., 2000). 

This is why we used a metagenomic sequencing approach—rather than cultures—to better 

understand the target microorganism community. It is important to understand which microbial 

guilds dominate these environments, how they live and interact with each other, and how 

selective environmental pressures influence these interactions.   

 

The environmental pressures of hot springs include a range of moderate to high temperatures, 

extreme pH ranges, and several toxic compounds. Mercury (Hg) is one such compound that has 

been observed at measurable concentrations in ~20% of geothermal sites in western North 

America (Geesey et al., 2016). Hg is a pollutant and a toxic metal present throughout many 

natural environments, such as hot springs and sediments. Due to its toxicity, Hg applies 

selective pressure on local organisms, especially in high concentrations. Mercury-resistant 

organisms can be found in some geothermal sites in North America, such as Yellowstone 

National Park (Geesey et al., 2016). Microbes that can tolerate high concentrations of Hg often 

have genes that allow them to reduce Hg(II) to its volatile form, Hg(0). Some prokaryotes have 

the mer operon, associated with mercury reduction, which encodes for mercury transport and 

transformations (Barkay and Wagner-Dobler, 2005). There are many different mer genes that 

participate in the uptake of Hg(II) into the cell. After being reduced, Hg(0) is released into the 

atmosphere in its gaseous state (Barkay and Wagner-Dobler, 2005). It is crucial to understand 

how organisms withstand toxic pollutants in extreme conditions within a properly functioning 

ecosystem.  

 

In this study, we aim to characterize the microbial community in Yellowstone National Park and 

identify the mechanisms of mercury resistance that allow them to survive in these geothermally 



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diverse hot springs. For this purpose, DNA sequencing was used to identify the mercury 

reducing organisms or mercury methylating organisms present in the community. In addition, 

diversity metrics were used to determine if the physical and chemical properties of the spring 

environment correlate with the structure and function of their microbial communities. Our study 

differs from previous literature in that we have a pH and temperature range as well as a matrix 

range. This allows us to study the differences between different types of samples taken from 

the same site, i.e. scum vs. sediment from the sample site. We used DNA sequencing and 

diversity metrics to broaden our understanding of the structure and function of the microbial 

community in the hot springs of Yellowstone National Park. This information can help us better 

understand the impact of microorganism communities and their diversity in nature.  

 

Methods 

  

Geochemistry 

 

The conditions of the hot springs (i.e. temperature and pH) from which the samples were 

extracted were recorded by Dr. JoAnn Holloway and team. 

 

Sample Description 

 

The samples were collected in Yellowstone National Park in September 2017 by Dr. JoAnn 

Holloway (USGS). Samples were taken from Upper Geyser Basin, Nymph basin, Norris Geyser 

Basin, Mud Volcano, Artist Paint Pots Basin, Gibbon Canyon, and Midway Geyser Basin. Samples 

were then stored in Lifeguard solution (Qiagen). For each sample, approximately one gram was 

collected and placed in a tube with Lifeguard solution. Scum and water samples were each 

collected with a sterile syringe and then filtered. Filters were placed into a Lifeguard solution 

and then stored on ice. Samples were shipped to Rutgers University and stored at -80°C until 

further analysis. 

 

Extraction 

 

DNA was extracted using the MoBio Powerlyzer PowerSoil DNA Isolation kit (Qiagen) following 

the manufacturer’s protocol. This kit is intended to be used for the isolation of microbial 

nucleic acids from soil, environmental samples, and tough microbes. It uses bead-based 

homogenizers to eliminate humic substances and other inhibitors, which allows for downstream 

application. DNA extracts were stored at -20°C following extraction.  

 

PCR 

 

PCR (Polymerase Chain Reaction) was performed after every extraction to amplify the 16S rRNA 

gene from the extracts to determine the presence of bacterial and/or archaeal DNA. For PCR 

products obtained with GoTaq Green Master Mix (Promega), a 515 forward primer and 806 



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reverse primer 16S V4 primer set was used (Parada et al., 2016; Apprill et al., 2015).  Products 

of PCR were separated by 1% agarose gel electrophoresis. 

 

DNA Sequencing 

 

The V4 region of the 16S rRNA gene was sequenced using the Illumina MiSeq platform following 

the Earth Microbiome Project at the Rutgers University Genome Cooperative (UL-Hasan et al., 

2019).  

 

hgcA Detection 

 

PCR was performed on every sample to see if hgcA genes were present within the extracted 

DNA. hgcA was targeted using a 261 forward primer and a 912 reverse primer set as previously 

published (Schaefer et al, 2014). Products of PCR were separated by 2% agarose gel 

electrophoresis.  

 

merA Detection 

 

PCR was performed on every sample to see if merA genes were present within the extracted 

DNA. The PCR products were obtained with GoTaq Green Master Mix, a merA2 forward primer 

and a merA2 reverse primer set was used (Poulain et al., 2015). Products of PCR were separated 

by 2% agarose gel electrophoresis.  

 

Microbial Ecology Analysis (Figure 1) 

 

The bioinformatics platform for Quantitative Insights Into Microbial Ecology 2 (QIIME2) was used 

because it includes sequence alignments, quality filtering, phylogeny building, and taxonomic 

classification (Hall et al., 2018). QIIME2, as opposed to QIIME, allows for microbial marker gene 

analysis to better quantify and compare the extracted genomic material (Hall et al., 2018). For 

data analysis, the Divisive Amplicon Denoising Algorithm 2 (DADA2) pipeline was used to detect 

and correct the processed sequences (Callahan et al., 2016). Alpha rarefaction and beta 

diversity metrics were all performed and analyzed (Anderson, 2001). Taxonomy was assigned 

using a naïve-Bayesian classifier trained on the V4 region of the Silva-132 99% 16S database 

(Pedregosa et al., 2011) Then, a feature table and a feature data summary were made after 

sequence filtering. Lastly, a taxonomic bar plot was created along with beta diversity metrics 

such as Weighted UniFrac, which qualitatively measures community dissimilarity (Bolyen et al., 

2019; Anderson, 2001).  



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Results 

 

Samples were taken from various geothermally diverse sediments, waters, mats, and scum 

sites. A total of twenty-two samples were taken from distinct environments that ranged in pH 

from 1.71 – 6.65 and in temperature from 54.2°C – 87.8°C. These twenty-two samples came 

from various springs, geysers, pools, and basins located in Yellowstone National Park (Figure 2). 

Several sample matrices were taken in duplicate. 

 



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After collection, the twenty-two samples were sent for sequencing analysis after completion of 

the genomic extractions. Eleven out of twenty-two samples were retained after rarefication at 

500 or more sequence reads (Table 1). The other eleven samples had very small amounts of 

biomass and thus resulted in a low number of reads (<500), so these samples were excluded 

from further analyses. The samples that had a low number of reads consisted mainly of water 

and scum samples from sample matrices.   

 



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Weighted UniFrac analysis was performed to observe how the various sample matrices cluster 

together based on several environmental factors that drive community selection. The three 

main selection factors that were investigated were temperature, pH, and sample matrix. These 

environmental conditions were tested to isolate the primary factor that influenced sample 

clustering. The Principle Coordinate Analysis (PCA) plot showed that the samples exhibited 

clustering based on sample matrix (Figure 3a). One cluster was composed of sediment and 

outlet sediment samples, while the other was composed of sediment and water samples. The 

mat and scum samples, both from Mud Volcano, did not cluster together with the other 

samples. In addition, the pool edge sediment from the Crystal Sister West sample was shown to 

be distinct from the other sample matrices.   

 

The Weighted UniFrac beta diversity analysis showed that the microbial communities in 

Yellowstone National Park clustered primarily based on temperature, rather than pH, which had 

no clustering pattern (Figures 3b and 3c). Microbial communities from samples that had a 

temperature of 50-60℃ tended to cluster together, while communities that were taken from 

sampling sites with a temperature of 70-90℃ tended to cluster together, with the exception of 

Crystal Sister West sediment. Beta diversity dissimilarity results showed that pH accounts for 

13.9% (p = 0.006) of variance, while temperature accounts for 22.7% (p < 0.001) and sample 

matrix accounts for 38.1% (p = 0.009).  

   



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Sequence taxonomy was assigned to determine the major taxa present in the samples. The 

archaeal order Thermoproteales was the most abundant throughout the samples, except for at 

the Mud Volcano mat and Nymph water sites, where it was not detected (Figure 4). The second 

most dominant order was Candidatus Micrarchaeum, which was present in seven of the 

samples. In addition, the most dominant bacterial taxon was Acetobacterales, while the most 

dominant archaeal taxon was Desulfurococcales. Overall, there were more archaea than bacteria 

present in physiologically extreme conditions.  

  



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Within the eleven samples, those that had more than a 50% relative frequency of archaea 

included Mud Volcano mat, scum, pool edge sediment, and water; sediments from site samples 

Crystal Sister E, Nymph, Crystal Sister W, Mud Volcano; and outlet sediments from Gibbon 

Canyon. The samples dominated by bacteria (more than 50% relative frequency) were Mud 

Volcano scum and mat. Within Mud Volcano, samples collected from the sediment were 

dominated by archaea, while the mat and scum samples were dominated by bacteria. 

Candidatus Micrarchaeum was abundant in samples with a pH range of 1.0-2.32. 

Desulfurococcales and Thermoproteales were present in the pH range of 1.71-6.65. Archaeal 

16S rRNA reads were detected in all samples with pH at or below 6.65.   

 

Lastly, all of the samples were also tested for mercury reduction or mercury methylation genes. 

The gene encoding a mercuric reductase, merA, was detected in two samples from Mustard 

Spring and one sample from Nymph. Mustard spring has a pH of 8.19 with a temperature of 

86.2 ºC, while Nymph Spring has a pH of 2.32 and a temperature of 59.5 ºC. No copies of hgcA, 

a gene responsible for Hg methylation, were detected in the samples.   

 



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Discussion/Conclusion 

 

Sequences of 16 rRNA gene PCR products were obtained from most of the samples that were 

extracted. Samples that had over one thousand sequence reads were mostly sediment samples. 

Fewer reads, above one hundred but below one thousand, were obtained from the mats, water, 

and scum samples. In addition, the samples excluded from the PCA plot had very low number 

of reads (<500 reads) such as mat and water samples. This is not that surprising because (i) 

samples were very small—yielding little biomass in all but the sediments and extreme biomass 

scarcity in water samples—and (ii) the DNA extraction kit was most suited for soil and sediment 

samples.  

 

The beta diversity analysis showed that the microbial communities in Yellowstone National Park 

cluster primarily by temperature rather than pH. This matches previous literature that states 

that the main selection factor in hot springs tends to be temperature (Uribe-Lorio et al., 2019). 

Beta diversity dissimilarity results showed statistical significance of sample separation based on 

pH, temperature, and sample type. The Weighted UniFrac PCA plot, which measures community 

dissimilarity while incorporating the relative abundance of the organisms, showed two distinct 

groups clustered based on the temperatures of the sites (Lozupone et al., 2011). In addition to 

these two grouping clusters, the results also showed a dominance of archaeal species. This is 

likely due to the extreme thermophilic and pH conditions which allow archaea to outcompete 

bacteria in these niches (Reed et al., 2013). Archaea are known extremophiles that have evolved 

specific proteins that allow them to proliferate in extreme environments and tolerate normally 

toxic compounds, such as arsenic and mercury (Reed et al., 2013). It is not surprising that they 

dominate these communities in the extreme conditions of some Yellowstone springs.  

 

In addition to temperature, the microbial taxa also clustered based on sampling matrix. 

Clusters for sediments and outlet sediments, pool edge sediments, water, and scum samples 

were all observed. The Weighted UniFrac plot showed two distinct clustering patterns between 

the different sample matrices. Environmental conditions in the different matrices are expected 

to vary. Sediments are expected to contain less dissolved oxygen, less UV radiation, more 

sulfide, more arsenic, and higher mercury concentrations compared to water, mat, and scum 

samples, which were taken from the top of the hot spring (Jiang et al., 2016).  

 

Sequence reads were taxonomically classified at the order level for all sites where more than 

500 sequence reads were available. Around 10% of the obtained reads were unidentified, 

possibly due to many microorganisms being unculturable and thus not being included in 

databases. Results showed that the archaeal order Thermoproteales was present in all but one, 

Crystal Sister West, of the sampling sites, indicating that this order thrives in these extreme 

thermophilic conditions. Since Thermoproteales was detected in most of the sequenced 

libraries, it can be inferred that this order is selected for in these niches, and that its growth is 

not limited to a singular matrix in the hot spring. This aligns with previous literature that 



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depicts diverse metabolic capabilities of Thermoproteales, which includes sulfur 

oxidation/reduction, oxygen respiration, H2 respiration, and CO2 fixation (Jay et al., 2016).  

 

Several samples from the Mud Volcano site were collected, extracted, and sequenced. These 

included three mat samples, one water sample, two pool edge sediment samples, and two scum 

samples. This allowed us to compare communities from different matrices and locations within 

one site. The Mud Volcano sediments (central and pool edge sediment) were dominated by 

archaea while the mats and scum showed the presence of both bacteria and archaea. Mud 

Volcano has moderately hot and acidic water (Table 1), which has magnesium, calcium, and 

sodium along with high concentrations of both organic carbon and sulfur (Sherman et al., 

2009). These are all necessary trace minerals that can be used for energy and growth, so it is 

not surprising that we found an abundance of both acidophilic and thermophilic bacteria and 

archaea.  

 

Observing the abundance of taxa in different sample matrices and springs suggests a potential 

gradient. Most of the samples dominated by archaea are the sediment samples, while the mats, 

water, and scum samples are dominated by bacteria (excluding Mud Volcano). The anoxic 

sediments of the hot springs, which have a higher concentration of Hg and sulfide (Krabbenhoft 

et al., 1999), show an abundance of archaeal species. On the other hand, the mat, water, and 

scum samples were taken from the water above the sediment and thus exposed to oxygen, 

sunlight, less mercury, and less sulfide has a higher abundance of bacterial taxa. This is likely 

due to the innate characteristics of archaea, such as a lipid monolayer and having specific 

proteins that have evolved to remain stable and active in various harsh conditions that allow 

them to thrive and proliferate in extreme environments (Reed et al., 2013). In addition, such 

gradients could be due to the competition between the archaea and bacteria. The archaea 

outnumber the bacteria at the bottom of the springs, thus out-competing them for resources 

and nutrients. Archaea are better adapted to survive in oligotrophic, low nutrient environments. 

They are able to live in sediments that are energy stressed due to having lower cell permeability 

than bacteria, thus reducing energy loss (Vuillemin et al., 2019). In addition, archaea are also 

able to outcompete bacteria for urea, which is used as a source of carbon for the cell (Seyler et 

al., 2019). This shows that archaea have developed several adaptations that allow them to 

utilize nutrients that often cannot be used by bacteria, thus allowing them to grow and survive 

in more environments and outcompete bacteria for the limited nutrients.  

 

At low temperatures, bacterial and archaeal species were found in similar abundances, while 

archaea were much more dominant at high temperatures. This indicates that higher 

temperatures allow archaeal dominance within acidic to neutral pH ranges (1.71-6.65). This 

might be because most alkaliphilic archaea are also considered halophilic due to sharing similar 

genomic characteristics (Reed et al., 2013). The sampled alkaline springs, like Octopus Spring, 

did not have a high salt concentration, thus possibly selecting hyperthermophilic and 

alkaliphilic bacteria instead of archaea.  

 



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In Yellowstone National Park, as well as other geothermal springs, there are often moderate to 

high levels of toxic compounds, such as Hg, or those that may serve as growth substrates, such 

as sulfur. Consequently, organisms often carry genes for resistance to these compounds or may 

utilize these compounds for better survival and growth. In our study, genes necessary for Hg 

methylation, hgcA, were not detected in any of the hot spring samples, and no known Hg 

methylating genera were detected by 16S sequencing. This, however, does not indicate that 

there is no presence of Hg methylation, it just means that the samples that were collected did 

not contain microorganisms with this gene or that organisms with this gene were detected but 

not characterized due to the limited amount of information that is known about uncultured 

extremophiles.  

 

When looking at Hg resistance, the taxonomy derived from the 16S sequences showed the 

presence of the archaeon Sulfolobus spp., which has a mer operon (Barkay and Wagner-Dobler, 

2005), in the Infant Geyser outlet sediment sample. This suggests that it is possible that Infant 

Geyser, which has a pH of 3.05, ~5.5 x 104 ng/L of total mercury, and a temperature of 82.7℃, 

has thermophilic mercury resistant species (Barkay, Personal Communication). Sequence reads 

in the order Aquificales, which also contains the mer operon, were detected in several sampling 

sites including Nymph (~1,000 ng/L total mercury), Crystal Sister West (>10,000 ng/L total 

mercury), Mud Volcano (1 x 104 - 1 x 105 ng/L total mercury), and Gibbon Canyon (>1,000 

ng/L total mercury) (Geesey et al., 2016; Barkay, Personal Communication). Aquificales bacteria 

were most abundant in Nymph and Mud Volcano. This suggests a potential of mercury 

reduction occurring within those environments, and this makes sense because these four sites 

all have a pH of around two. We detected the merA gene in DNA from three samples from 

Mustard Spring and Nymph. Mustard spring has a pH of 8.19 with a temperature of 86.2 ºC 

while Nymph Spring has a pH of 2.32 and a temperature of 59.5 ºC. This data demonstrates 

that in the springs where the total mercury concentration in unfiltered water is around 1,000 

ng/L and above, there are taxa that have the potential for mercury resistance (Barkay, Personal 

Communication). In hot springs that have a low pH, there is often a higher concentration of 

total mercury, thus selecting for mercury resistant organisms. While the genes needed for 

mercuric reduction were detected within the samples, detection of the gene does not indicate 

activity. Further testing is needed to determine if mercury reduction genes are active within 

these species.   

 

It is important to understand Hg reduction because it plays a critical role in the global cycling of 

Hg. The reduction of mercury is the only transformation of a toxic metal that can be done on a 

large scale (Barkay et al., 2003). Microorganisms that are capable of mercuric reduction are able 

to transform both organic and inorganic Hg during biotic and abiotic processes (Barkay et al., 

2003). This transformation helps to prevent Hg bioaccumulation, which is a serious problem in 

some parts of the world. For many decades now, Hg reducing bacteria have been utilized by 

humans to remove toxins from human products such as wastewater (Barkay et al., 2003). This 

means that Hg reduction is not only necessary for ecosystem homeostasis, but is also 

fundamental in the reduction of Hg through bioremediation, which is the degradation of 



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environmental contaminants through the use of organisms, mainly microorganisms (Vidali, 

2001). The different taxa shown here to live in hot springs with high mercury concentration, 

such as Sulfolobus spp. and the Aquificales bacterial order, can be used for further research to 

support the bioremediation process.  

 

Our results correspond with previous literature about community structure within hot springs in 

Yellowstone National Park. Extracting metagenomic information from several springs 

demonstrated that the microbial communities cluster together based on the temperature of the 

spring and sample matrix. As one selection factor often dominates in extreme environments 

(Uribe-Lorio et al., 2019), it is important to know that the main selection factor for these 

communities in Yellowstone is temperature. A potential gradient based on the abundance of 

archaeal and bacterial species was observed in samples from different compartments within the 

same spring. Archaeal species were more abundant in the sediment samples and their presence 

decreased at the surface of the water, while bacterial species exhibited a reverse gradient where 

they were most abundant in the water and mats at the top of the spring and less abundant 

down towards the sediment. This shows that there is a negative correlation between the 

presence of bacterial and archaeal growth within the hot springs. This is likely due to the 

different environmental conditions and compounds that are present at the top and bottom of 

the hot spring. This information gives us a better understanding of how microbial communities 

form, function, and interact with the surrounding environment.  

 

Even though the data obtained in this study matches the previous literature, there are still 

several limitations that need to be addressed. First, the sample size of this study is small, so 

the data presented in this paper cannot be widely generalized. There were originally twenty-two 

samples, and only eleven of them had over 500 sequences and were kept for further analyses. 

Five different basins were sampled. All sites had at least one water sample, however, only six 

sites had mat samples and only five sites had sediment samples. It was hard to extract genetic 

material from the water samples, as they had very low biomass. Furthermore, in those water 

samples, even if material was extracted, the sequence number was low. Therefore, the samples 

could not be considered. This issue caused the number of usable samples for metagenomic 

analysis to decrease by half. Out of the five basins originally sampled, only four (Mud Volcano, 

Nymph, Gibbon Canyon, and Norris Geyser Basin) had samples that were used for analysis and 

data extrapolation. Another limitation is that some of the basins used for data analysis are 

similar in their pH and temperature range. This means that this data cannot be applied broadly; 

more samples from other basins with varying pH levels must be analyzed before results can be 

generalized. Additionally, this paper used a metagenomic approach to show that mercury 

reduction genes are present, while mercury methylation genes were not. This, however, does 

not indicate if the merA genes are active within the community. Rather, it only shows that these 

genes are present. Regarding the absence of mercury methylation genes, this might not be 

representative of all of the sites due to the small sample size that was analyzed. In addition, the 

Hg concentration was not measured at the time of sampling but rather at another time in the 

same year. Overall, one of the more considerable limitations to this study is that many of the 



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microorganisms tested were unculturable. This means that there is often little information on 

these organisms, so it would be impossible to classify genes even if they were detected. This 

happened during the taxonomic analysis of the eleven samples, which were often categorized 

with only domain, family classification, or just “uncultured.” These samples therefore did not 

have enough information for us to determine a bigger picture of the community structure.   

 

Due to the various limitations that are present in this study, there is more that can be done for 

future research and analysis. First, more samples need to be taken, specifically more sediment 

samples, and they should be taken from a wider range of basins located throughout 

Yellowstone National Park. This will not only help to increase the sample size but also to search 

for other mercury reducers and mercury methylators within the different environments. Second, 

other mer operon genes can be tested instead of just mercuric reductase (merA). This can 

improve understanding of the different capabilities of the microorganisms inhabiting hot 

springs. Lastly, proteomics analyses can be done to see if the merA genes are active within the 

sampled microorganisms. Other research that can further this study is to look at other ways 

these microorganisms resist toxic mercury concentrations. While some microbes do not have 

the mer operon, they could have other ways to resist Hg. These methods could include influx, 

accumulation, efflux, and metallothionein, a protein that protects the cell against the toxicity of 

Hg (Irawati et al., 2012). It is important to know the many diverse ways that microorganisms 

resist toxic metals. This new information can give us a more holistic view of the unique 

environment of the microorganisms in the hot springs of Yellowstone National Park.  

 

Acknowledgments 

 

We would like to thank Dr. JoAnn Holloway and her team for collecting all of the samples in 

Yellowstone National Park.  

 

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