Diversity and Distribution of sulfate-reducing …Many bacteria can use sulfur compounds in...

Marine Biological Laboratory Semester in Environmental Science 2011

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Diversity and Distribution of sulfate-reducing bacteria in Siders Pond,

a meromictic salt pond

Sarah Nalven

Department of Biology, Colby College, Waterville, ME 04901 USA

Abstract Genetic diversity and community structure of sulfate-reducing bacteria was investigated down

the 12 meter water column of Siders Pond, a meromictic salt pond in Falmouth, MA. The physical and

chemical gradients down this water column were also investigated, as they provide distinct niches for

the microorganisms of Siders Pond. Microbial DNA was extracted from water samples at 12 depths. The

dissimilatory sulfite reductase (dsr) gene was amplified via PCR, and dsr amplicons from three depths

were subsequently cloned and sequenced. Of the 12 sampled depths, dsr was detected in the lower 10

samples, all of which were taken from the anoxic portion of the water column. The dsr sequences in 12

m were genetically distinct from the dsr found in the 4 and 8 m samples, and contained more diversity,

exemplifying niche differentiation of sulfate-reducing bacteria along the profile of the water column.

Using BLAST to compare Siders Pond dsr sequences to the GenBank database also showed that the

closest relatives of Siders Pond sulfate-reducing bacteria were all isolated from human-impacted and

organic-rich environments.

Key words: sulfate-reducing bacteria, dissimilatory sulfite reductase (dsr), meromictic, water column,

pelagic zone, diversity, distribution, niche differentiation.

Introduction Many bacteria can use sulfur compounds in energy-yielding reactions, called dissimilatory sulfur

metabolism. In one type of these reactions, sulfate (SO42-) is used as a terminal electron acceptor and

reduced to sulfide (S2-), while organic carbon compounds (such as organic acids, fatty acids or alcohols)

act as electron donors. Known as sulfate reduction, this process is strictly anaerobic and is carried out in

anoxic, sulfate-rich environments such as marine sediments, benthic microbial mats, and water columns

according to this equation:

CH3COO- + SO42- + H+ 2HCO3

- + H2S

Bacterial sulfate reduction is performed by a diverse set of organisms within proteobacteria, and

is essential to the global sulfur cycle and several other global cycles, including those of carbon, nitrogen

and phosphorus. In fact, sulfate reduction is the dominant carbon mineralization process in most

marine and coastal sediments (Jorgensen, 1982; Skyring, 1987). Although it is recognized that sulfate-

reducing bacteria play an important role in ecosystems and in global cycles, little is known about the

Diversity and distribution of sulfate-reducing bacteria

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genetic distribution of their populations, especially in water columns (Jorgensen et al., 1979; Overmann,

1996).

This study examined the water column of Siders Pond (Figure 1), a coastal pond in a residential

neighborhood of Falmouth, MA. The pond has a history of slight eutrophication (Moore 2009,

Unpublished Report), since Falmouth’s wastewater was not sewered until 1986 and until then

wastewater reached the pond through groundwater input. Today, the pond is completely surrounded

by houses, many of which fertilize their lawns, so nutrient loading still occurs. Trees also grow near the

shore of the pond, so the water receives some terrestrial carbon input.

Siders Pond is also meromictic, or never mixes below a few meters. This feature is mostly due to

the salinity of the water, which can be attributed to occasional seawater intrusion through a small

channel that connects the pond to Martha’s Vineyard Sound during large storms, but it is also due to the

shape of the pond’s basin, which is unusually deep for the pond’s surface area (Figure 2). Due to the

meromictic nature of the pond, the water column is stratified and steep physico-chemical gradients

have developed. These vertical gradients provide different habitats for the development of different

microbial communities within short distances. This means that communities of sulfate-reducing

bacteria living in different regions of the water column will have very different habitats, and will most

likely adapt to those habitats in a process called niche differentiation.

In order to examine niche differentiation in the water column of Siders Pond, a highly conserved

region of the dissimilatory sulfite reductase (dsr) gene was examined. Dsr encodes the enzyme

responsible for catalyzing the final step of dissimilatory sulfate metabolism, the reduction of sulfite

(SO32-) to sulfide (S2-). Through PCR, cloning and sequencing of dsr DNA isolated from several depths, as

well as characterization of the physico-chemical properties of the water column, diversity and

distribution of sulfate-reducing bacteria was evaluated.

Methods

Sampling. Water samples were collected on November 14, 2011 from Siders Pond in Falmouth, MA.

Samples were obtained in the southwest part of the pond where the water was 12.2 meters deep.

Samples for DNA analysis were collected at 2, 4, 4.5, 5, 5.5, 6, 7, 8, 9, 10, 11, and 12 m and stored in 1-

liter NalgeneTM bottles. Samples for nutrient analysis and cell counts were taken at every meter from 1

to 12 m depth and stored in 125-mL NalgeneTM bottles. Temperature, salinity, dissolved oxygen, and pH

were also measured with a Hydrolab sonde, DS5X (Hatch Hydrolab, Loveland, CO), at 1, 2, 3, 4, 4.5, 5,

5.5, 6, 7, 8, 9, 10, 11, and 12 m.

Inorganic Nutrient Analysis and Cell Counts. Samples were analyzed for sulfate (SO42-) with an ion

chromatograph, DX-120 (Dionex, Sunnyvale, CA) and for sulfide (S2-) with a UV colorimetric

spectrophotometer, UV-1601 (Shimadzu, Kyoto, Japan). Expected sulfate was calculated by multiplying

measured salinity values by 806.5, a known seawater salinity to sulfate ratio. Cells were enumerated

using 4',6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy.

DNA Isolation. Pond water was filtered from each depth onto 0.22-μm-pore-size polyethersulfone SterivexTM filter units using a gentle vacuum (Figure 3 for filtering set-up). Different volumes of water

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were filtered for each sample based on ease of filtration (Table 1). Filters were stored at - 80°C until DNA extraction. DNA was extracted from each of the 12 samples using the MOBIO PowerWater® SterivexTM DNA Isolation Kit. Samples were then run on a 1% agarose gel and visualized with ethidium bromide to confirm successful extraction. Extracted DNA was also quantified using a Micro-Volume UV-Vis Spectrophotometer, NanoDrop 2000 (Thermo Scientific, Waltham, MA).

PCR Amplification. Dsr DNA sequences were amplified from each sample of isolated DNA with the

primers dsr1F and 1334R (Santillano, 2010). Total volume of the PCR amplification reaction was 50 μl,

which included 1 μl of template DNA, 5 μl of each primer (20 μM), 1 μl of deoxynucleoside triphosphates

(10 mM), 2 U Taq polymerase, 10 μl of 5x PCR buffer, and 27.8 μl of sterile water. The amplification

conditions involved one step at 95°C for 3 min., then 35 cycles at 94° for 35 sec., 54°C for 1 min., and

72°C for 2 min., and then a last step of 72°C for 10 min. PCR-amplified DNA was run on a 1% agarose gel

and visualized with ethidium bromide to determine whether the dsr gene was present. DNA from

depths 4.5, 8, and 12 m were then amplified in triplicate, and products were pooled prior to further use.

Cloning. Pooled PCR products from 4.5, 8, and 12 m were purified using the QIAquick PCR Purification Kit. Each purified product was then separated on a 0.8% agarose gel, and 900 bp bands were excised and re-purified using the QIAquick Gel Extraction Kit. PCR-amplified dsr DNA sequences from the three depths were then ligated into the pCR™4-TOPO® TA vector and transformed into Electrocomp™ E. coli using the PureLink™ Quick Plasmid Miniprep Kit (Invitrogen, Grand Island, NY) and an electroporator at 1600 V, Eppendorf Multiporator® (Eppendorf, Hamburg, Germany). Clones containing dsr from each of the depths were then grown in Super Broth to prepare for sequencing.

DNA Sequencing. Each clone underwent plasmid isolation (alkaline lysis) and bidirectional sequencing,

ABI v 3.1 BigDye terminator (Applied Biosystems, Carlsbad, CA) at the Keck Facility of the JBPC, MBL,

Woods Hole, Massachusetts. The dsr gene was sequenced from 37 different 4.5 m clones, 40 different 8

m clones, and 38 different 12 m clones for a total of 115 dsr sequences.

Phylogenetic Analyses. Sequences were aligned so a distance matrix could be calculated. Using this

matrix, sequences were clustered into operational taxonomic units (OTUs) with the software MOTHUR.

Several sequence difference levels were calculated, but an 8 percent sequence difference level was

chosen to cluster the sequences into 17 OTU representative sequences for further phylogenetic analysis.

The Bray Curtis similarity index was used to compare OTU sequences with each other and BLAST was

used to compare OTU sequences against the GenBank dsr database.

To relate each of these OTUs to each other and to similar sequences found in GenBank, a phylogenetic

tree was created. Using NCBI’s Batch Entrez, accession numbers were entered and a fasta file was

downloaded. This file was opened in BioEdit, sequences were aligned and trimmed, and files were finally

entered into Phylogeny.fr: Robust Phylogenetic Analysis for the Non-Specialist in order to create a tree.

FigTree was used to enhance tree design.


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Results

Characterizing the water column. Saline stratification is evident down the water column, as sharp

increases in salinity are seen at about 3 m and 8 m depth, dividing the water column into three distinct

layers (Figure 4). All physical parameters are completely uniform throughout the top layer of the water

column, which extends down about 3 m. At 3 m, a sharp decline in dissolved oxygen begins until the

water column becomes completely anoxic at 6 m. Temperature in the surface layer is about 11.3 °C,

slightly warmer than 10.2 °C, the average atmospheric temperature of the two weeks prior to sampling.

The middle layer reaches temperatures over 17 °C, and the bottom layer, like the surface, is about 11 °C.

Water at the surface is slightly alkaline, but a substantial decrease in pH exists at the oxycline from

about 3 to 5 m, making the water slightly acidic by 4 m depth (Figure 5). At 5 m, this decreasing trend in

pH turns around, but it is not until 6 or 7 m that pH increases substantially. By 9 m, the water column is

alkaline once again, and continues increasing for the remaining measurements.

The chemical composition of the water column also indicates stratification (Figure 6). Because expected

sulfate was calculated based on measured salinity values, the trend in expected sulfate shows the same

three layers as salinity does, and increases down the water column. Measured sulfate follows the same

trend as expected sulfate in the upper section of the water column. However at 7 m the discrepancy

between expected and measured sulfate begins to widen, and continues to widen until the bottom of

the water column. (The oscillation seen in the measured sulfate values toward the bottom can be

attributed to noise in the measuring methods.) Also at 7 m, concentrations of sulfide become

detectable and continue to increase down the water column.

Samples from 1 and 2 m depth contained 106 cells/mL while all samples from 3 m and below contained

107 cells/mL. Cell concentrations were highest at the oxycline and at the bottom of the water column

(Figure 7). Observations of cells under the microscope also revealed that cell structure was much more

varied higher up in the water column and ranged in size, shape, and color, while cells were much all

small and uniform further down in the water column (Figure 8).

DNA Isolation and PCR. As the cells that were once suspended in each depth of the water column were

trapped onto each filter, filters turned unique colors (Table 1, Figure 9). DNA was successfully isolated

from each of these filters, and each samples produced a different concentration of DNA (Table 1). When

each of these DNA samples underwent PCR and gel electrophoresis, the 2 m and 4 m samples showed

extremely weak or nonexistent dsr signals, but every sample from 4.5 m and below showed strong dsr

signals (Table 2).

DNA Sequencing and Phylogenetic Analyses. Dsr DNA was successfully sequenced from all three

samples (4.5, 8 and 12 m). Of the 115 sequences, 106 were unique; at the 8 percent distance level,

sequences were clustered into 17 OTU sequences (Table 3). Of the 17 OTU sequences, certain

sequences were very abundant at one or two levels of the water column, but not one sequence was

isolated from all three levels of the water column (Table 4).

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The most OTU sequences were isolated from the 12 m sample (12 sequences), while the fewest

sequences were isolated from the 8 m sample (3 sequences) (Figure 10, Table 4). The 4.5 m sample

shared sequences with both the 8 m sample and the 12 m sample, but the 8 m and 12 m sample did not

contain any of the same OTU sequences.

The Bray Curtis similarity index determined that sequences from 12 m were quite genetically distinct

from the other two layers (Figure 11). While the sequences isolated from 4.5 and 8 m were about 80

percent similar, they were each only about 4 percent similar to the sequences isolated from 12 m.

Lastly, based on similarity of dsr sequences, BLAST found the closest known relatives of the Siders Pond

sulfate-reducing bacteria on the GenBank database (Figure 12). Certain sequences were closely related

to known cultured organisms such as Desulfovibrio aminiophilus, Desulfococcus oleovorans, Desulfospira

joergensenii, Desulfobaccula phenolica, and both naphthalene and phenanthrine-mineralizing sulfate-

reducing bacteria.

OTU 4 was most abundant in the water column and was isolated 28 times from 4.5 m and 34 times from

8 m, but not once from 12 m (Table 4). OTU 4’s closest relative in Siders Pond was OTU 2 which was

found 3 times in 4.5 m, but not once in the other layers (Figure 12, Table 4). Both sequences were found

to be most similar to a sequence isolated from the sludge of a marine aquaculture biofilter when

BLASTed against the GenBank database (Figure 12).

OTUs 6, 7, 8, 9, 10, and 11 constituted a cluster on the phylogenetic tree and were all found only or

mostly in 12 m (Figure 13). This cluster is related to a known sulfate-reducing, phenanthrine-

mineralizing bacterium isolated from Arthur Kill, NY (Perez-Jimenez, 2000).

Discussion

Characterizing the water column. Salinity change is the driving force behind the meromictic nature of

Siders Pond—the saltiest, densest water is found at the bottom of the water column, and the least salty,

least dense water is found at the top. The top layer of the water column is clearly mixed by the wind

since all physical parameters are uniform above approximately 3 m depth. Because of its contact with

the atmosphere, the mixed layer also has much higher concentrations of dissolved oxygen than the rest

of the water column, which is completely depleted of oxygen by about 6 m. During the fall when the

atmosphere cools, this contact also causes the water’s heat obtained during the summer to diffuse out

of the mixed layer. This is why the water of the middle layer has much higher temperatures than the

top layer: it has much less contact with the atmosphere and is therefore able to retain summer heat for

longer.

The variation in several chemical parameters down the water column suggests that different

communities of bacteria occupy different niches down the water column and influence the

biogeochemistry of their respective niches. Sulfate-reducing bacteria probably have a large influence on

the biogeochemistry of the lower portion of the water column. When pH drops below the mixed layer

at the oxycline, it is most likely due to several types of bacteria that oxidize the chemicals in this layer,


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(including hydrogen sulfide) and produce hydrogen ions in the process. Since sulfate-reducing bacteria

use a hydrogen ion in the reduction of sulfate, however, these bacteria would influence pH in the other

direction and cause pH of the water column to increase. Not surprisingly, the pH of the water column

begins to increase at 4.5 m, and continues to increase down the remainder of the water column, the

exact zone where the dsr gene was detected. Furthermore, the water becomes much less acidic at 7 m,

exactly where hydrogen sulfide becomes apparent in substantial concentrations. Presumably sulfate-

reducing bacteria are removing hydrogen ions from their surroundings and turning them into this

hydrogen sulfide.

Comparison of expected to measured sulfate values further suggests presence of sulfate-reducing

bacteria in the lower portion of the water column. The apparent depletion of sulfate in the water

column below about 7 m (according to calculated expected sulfate values) indicates that sulfate-

reducing bacteria are the cause of this depletion as they reduce sulfate. The detection of hydrogen

sulfide production and sulfate depletion at about 7 m however does not mean sulfate-reducing bacteria

are not active until this point: higher in the water column, hydrogen sulfide produced could be getting

oxidized by other organisms, and sulfate depleted could be overshadowed by sulfate produced,

therefore preventing detection.

Cell abundance most likely increases at the oxycline due to the many microhabitats the oxycline

provides. Once oxygen is no longer available as an electron acceptor, organisms capable of respiring

other compounds can occupy different niches according to the electron acceptors present in the water

column at that point and their energetic properties. Galand et al. found that in the Clipperton Lagoon

(California), prokaryotic cells were four times more abundant in the oxycline than in the rest of the

water column, and putative green sulfur bacteria represented half of these cells (2011). This could be

the case in Siders Pond as well. The increase in cell abundance at the bottom of the water column could

be also be attributed to sediment influence, which might increase the number of microhabitats and

provide plenty of organic matter for the bacteria to mineralize. Cell count could also increase due to the

absence of grazers since toxic levels of sulfide and other chemicals might prevent grazers from living in

the lower portion of the water column. Joye et al. (1995) and Casamayor et al. (2002) found that sulfide

and ammonia toxicity may result in the exclusion of zooplankton grazers from anoxic or hypoxic regions

of the water column. The absence of grazers could also explain the difference in the microscope

observations between samples from the top and bottom of the water column. Cells in the upper layers

were probably more varied in shapes and sizes because some were phytoplankton cells or grazers. In

the lower layers, however, phytoplankton can no longer survive because there is no light, and grazers

can no longer survive due to toxicity of certain chemicals.

DNA Isolation and PCR. Detection of the dsr gene in every sample from 4.5 m and below indicates that

sulfate-reducing bacteria are present in the anoxic region of the water column. The ambiguous signal

from the 2 and 4 m samples should be studied further in order to determine whether dsr was in fact

present. If it was, its presence could be attributed to slight mixing between the water at 4.5 m and

upper layers. It is also noteworthy that detection of dsr DNA only suggests presence of sulfate-reducing

bacteria, and it does not suggest activity; even if sulfate-reducing bacteria were in fact present in the

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upper part of the water column, they might not necessarily be active. It is also possible that sulfate-

reducing bacteria present in the upper part of the water column were active, but they had undergone a

metabolic switch, in which they used a more energetically favorable electron acceptor such as nitrate

(NO3-). Seitz & Cypionka have found that some organisms are capable of this metabolic switch (1986).

DNA Sequencing and Phylogenetic Analyses. Sequencing of dsr genes from the water column of Siders

Pond revealed the presence of novel dsr sequences, which is to be expected considering how rapid

microbial turnover and therefore microbial evolution is, and also considering how few sulfate-reducing

bacteria have been cultured or identified. Sequencing also revealed that the Siders Pond water column

is not simply colonized by a few sulfate-reducing organisms, but that there are many sulfate-reducing

communities present, and the distribution of these communities demonstrates structure.

Community structure is known because each OTU sequence was abundant at only one depth or at two

adjacent depths (Table 4). In fact there was quite a bit of community overlap between 4.5 and 8 m since

these samples shared two abundant OTU sequences (Figure 10), including the most abundant sequence,

OTU 4. Considering the physical profile of the water column, however, it is not surprising that 4.5 and 8

m contain so many common sequences since the physical characteristics at the two depths are not

drastically different (Figure 4): 4.5 m is at the very top of the water column’s middle layer and 8 m is at

the very bottom, but both physically and chemically these two habitats are not nearly as different from

each other as they are from 12 m. It is also possible that since the two depths are found in water of

similar densities, there is some gene flow between the depths.

The sequences found in 12 m were quite distinct from the sequences found from either of the depths

above 12 m (Figure 11), which is probably because the water at 12 m has very different physico-

chemical characteristics from the other layers (Figures 4, 5 and 6), causing natural selection for different

dsr genes in 12 m than in the other layers. It is likely that the 12 m sequences were also distinct because

they were extremely isolated from the other levels of water and had been for a long time, due to salinity

and therefore density differences, and due to the lack of wind-driven mixing or bioturbation in the

bottom layer. Isolation would have allowed deep evolutionary branching of organisms in 12 m without

any gene flow from the layers above.

This would also explain why the sequences isolated from 12 m were not only genetically distinct (Figure

11) from the 4.5 and 8 m sequences, but why they were much more genetically diverse (Figures 10 and

12). In addition to isolation, elevated diversity at 12 m could also be attributed to the extreme

environmental conditions at this depth. Compared to the rest of the water column, 12 m contained the

highest salinity and pressure, as well as the highest concentrations of sulfide and possibly other

chemicals that have accumulated over the pond’s meromictic history. Galand et al. (2011) and

Humayoun et al. (2003) both also found the meromictic bottom waters of Clipperton lagoon and Mono

Lake, respectively, to have the highest microbial diversity. Galand et al. proposed that the unique

conditions of the deep layers of the Clipperton lagoon may explain the novelty of the microbial

communities found there, which were characterized by anoxia, high concentrations of organic carbon,

inorganic nitrogen compounds, an iron. Humayoun et al. also proposed that in Mono Lake the extreme


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bottom water, characterized by sulfide and ammonia toxicity, may have contributed to elevated

diversity (2003).

One final explanation for elevated diversity at 12 m is that dsr genes here were influenced by the

sediment, since the water column sampled was about 12.2 m deep. However it is not likely that the

sediment would be suspended 0.2 meters up the water column since there is no wind-driven current at

12 m or bioturbation.

Creation of a phylogenetic tree that included Siders Pond dsr sequences and their closest relatives from

GenBank allowed insight into evolutionary relationships amongst the Siders Pond sequences and the

physiological properties of the sulfate-reducing bacteria to whom they belong. Several sequences that

were isolated only from or mainly from 12 m constituted a cluster in the phylogenetic tree (Figure 13).

The organisms in this cluster were related to a known bacterium that mineralizes phenanthrine and was

isolated from Arthur Kill, NY, a hydrocarbon impacted waterway (Perez-Jimenez et al. 2000). This

suggests that the environment of the water column at 12 m could contain phenanthrine, and is most

likely hydrocarbon-rich, like the environment from which the known phenanthrine-mineralizing

bacterium was isolated. Several other similar sequences were also isolated from hydro-carbon rich

environments, many of which were also human-impacted. It is logical that Siders Pond would be

hydrocarbon rich because it is eutrophic. Some hydrocarbons in the water column could also be coming

from sinking terrestrial input, though further investigation would be required to determine this

conclusively. Regardless, the fact that these sulfate-reducing bacteria in Siders Pond are similar to

organisms that were investigated for bioremediation purposes suggests that the sulfate-reducing

bacteria that exist at 12 m in Siders Pond could have the potential to degrade petroleum or other

pollutants, or could even be doing so in Siders Pond currently.

OTU sequence 4, the most abundant sequence isolated from the water column, was found in both 4.5

and 8 m about 30 times, but was not closely related to any sulfate-reducing bacterium with known

physiological properties. Its closest relatives were organisms isolated from a marine aquaculture

biofilter (Schwermer et al. 2010), from groundwater contaminated by landfill leachate (Wu et al. 2009),

and from a New England salt marsh sediment (Bahr et al. 2004). Again, the environments from which

these similar sequences were isolated suggest the environment of Siders Pond that these sulfate-

reducing bacteria are living in, which seems to be polluted and organic-rich.

Finally, one interesting finding was that three different OTU sequences (OTUs 9, 11, 17) were found in

both 4.5 and 12 m, but never in 8 m. Furthermore, not one sequence was found in both 8 and 12 m.

One possible explanation for this finding is that sinking zooplankton fecal pellets may have transported

4.5 m sulfate-reducing bacteria into the 12 m region of the water column. Since there are probably not

many zooplankton at 8 m due to hydrogen sulfide toxicity, it would make sense that no sequence was

transported in this manner from 8 m to 12 m. In fact, Humayoun et al. proposed that in the meromictic

Mono Lake (California), sinking fecal pellets of the brine shrimp Artemia monica, the dominant

zooplankter, could have been responsible for transporting sequences from the top layer to the bottom

layer (2003). Another possible explanation is that in 4.5 m, these organisms are present as rare dormant

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cells, but in deeper waters, these cells develop as conditions become adequate. OTU 9, for example,

was found once at 4.5 m but 10 times at 12 m, and could exhibit this phenomenon. Galand et al.

proposes that this phenomenon could be responsible for the dispersal of deep water or marine

sediment organisms (2011). Lastly, 4.5 and 12 m both contain many more cells than 8 m (Figure 7), and

it could be that these particular dsr sequences somehow give sulfate-reducing bacteria an advantage in

waters with high bacterial abundance.

Conclusions

Sequencing and phylogenetic analysis of dsr in the Siders Pond water column, in parallel with a

physico-chemical evaluation of the water column, can tell a lot of information about the communities of

sulfate-reducing bacteria and the environments of their habitats. This study determined that sulfate-

reducing bacteria are present throughout the entire anoxic portion of the Siders Pond water column and

that these sulfate-reducing communities exhibit niche differentiation in their respective water column

habitats. Of the 4.5, 8, and 12 m dsr samples sequenced, 12 m dsr exhibited the most genetic distinction

and diversity. Similar dsr sequences from GenBank suggested that sulfate-reducing bacteria in every

layer of the Siders Pond water column have adapted to high concentrations of hydrocarbons and

possibly other pollutants.

Directions for Future Research

To continue this study, a more comprehensive assessment of the physico-chemical properties of

the water column would have to be done, especially an assessment of the types of hydro-carbons and

metals present. Activity of sulfate-reducing bacteria could also be examined since detection of dsr DNA

only suggests presence and not activity. Furthermore, attempts at culturing some of the sulfate-

reducing bacteria from Siders Pond could be carried out, especially OTU 4, the most abundant sulfate-

reducing bacteria isolated.

Acknowledgements

Many thanks to Julie Huber, my advisor; without her guidance this project could not have been

completed, nor would I have learned nearly as much. All the personnel in Julie’s lab were also of

tremendous help in teaching me several new molecular techniques and exhibiting the utmost patience

when I didn’t get things right the first time around. Special thanks to Angus Angermeyer and Emily

Reddington for taking hours out of their days to help me. Leslie Murphy also lent me her filtering rig,

and put up with the strong sulfidic odors of some of my water samples, which quickly pervaded the

entire lab. I would also like to thank all the Semester in Environmental Science (SES) teaching assistants,

especially Rich McHorney for critical assistance in the field and Carrie Harris and Stef Strebel for the

countless questions they answered and their assistance in lab. Also I would like to thank my secondary


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advisor, Joe Vallino, for critical assistance in the planning stages of this project and for making me

excited about microbes. Finally I would like to thank the Marine Biological Laboratory and SES for the

necessary funding for this project.

Literature Cited

Bahr, Michele, Byron C. Crump, Vanja Klepac-Ceraj, Andreas Teske, Mitchell L. Sogin, and John E.

Hobbie. "Molecular Characterization of Sulfate-reducing Bacteria in a New England Salt Marsh."

Environmental Microbiology 7.8 (2005): 1175-185. Print.

Casamayor, Emilio O., Ramon Massana, Susana Benlloch, Lise Øvreås, Beatriz Díez, Victoria J. Goddard,

and Josep M. Gasol. "Changes in Archaeal, Bacterial and Eukaryal Assemblages along a Salinity Gradient

by Comparison of Genetic Fingerprinting Methods in a Multipond Solar Saltern." Environmental

Microbiology 4.6 (2002): 338-48. Print.

Jørgensen et al., 1979 B.B Jørgensen, J.G Kuenen and Y Cohen, Microbial transformations of sulfur

compounds in a stratified lake (Solar Lake, Sinai). Limnol. Oceanogr., 24 (1979), pp. 799–822.

Jorgensen, Bo Barker. "Mineralization of Organic Matter in the Sea Bed-- the Role of Sulphate

Reduction." Nature 296 (1982): 643-45. Print.

Joye, Samantha B., Tracy L. Connell, Laurence G. Miller, Ronald S. Oremland, and Robert S. Jellison.

"Oxidation of Ammonia and Methane in an Alkaline, Saline Lake." American Society of Limnology and

Oceanography 44 (1999): 178-88. Print.

Galand, Pierre E., Muriel Bourrain, Emmanuel De Maistre, Philippe Catala, Yves Desdevises, Hila Elifantz,

David Kirchman, and Philippe Lebaron. "Phylogenetic and Functional Diversity of Bacteria and Archaea in

a Unique Stratified Lagoon, the Clipperton Atoll (N Pacific)." FEMS Microbiology Ecology 79 (2011): 203-

17. Print.

Humayoun, Shaheen B., Nasreen Bano, and James T. Hollibaugh. "Depth Distribution of Microbial

Diversity in Mono Lake, a Meromictic Soda Lake in California." Applied and Environmental Microbiology

69.2 (2003): 1030-042. Print.

McIntyre, A. D. "A Global Census of Marine Microbes." Life in the World's Oceans: Diversity, Distribution,

and Abundance. Ames, IA: Blackwell Pub., 2010. Print.

Moore, Marshall. “Getting to the ‘Core’ of Coastal Ponds: A Reconstruction of Biogeochemical Cycling

and Community Structure in Siders and Salt Ponds, MA.” SES Unpublished Report. 2009.

Overmann, Jorg, J. Thomas Beatty, H. Roy Krouse, and Ken J. Hall. "The Sulfur Cycle in the Chemocline of

a Meromictic Salt Lake." Limnology and Oceanography 41.1 (1996): 147-56. Print.

http://www.sciencedirect.com/science/article/pii/S0304420300000918#bBIB35

Sarah Nalven

11

Pennisi, Elizabeth. "How a Marine Bacterium Adapts to Multiple Environments." Science 24 Mar. 2006:

1697. Print.

Perez-Jimenez, J. R., L. Y. Young, and L. J. Kerkhof. "Molecular Characterization of Sulfate-reducing

Bacteria in Anaerobic Hydrocarbon-degrading Consortia and Pure Cultures Using the Dissimilatory

Sulfite Reductase (dsrAB) Genes." FEMS Microbiology Ecology 35 (2001): 145-50. Print.

Santillano, Daniel, Antje Boetius, and Alban Ramette. "Improved Dsr-A-Based Terminal Restriction

Fragment Length Polymorphism Analysis of Sulfate-Reducing Bacteria." Applied and Environmental

Microbiology 76.15 (2010): 5308-311. Print.

Schuler, Dirk. "2.1 Salt Marshes and Salt Ponds." Magnetoreception and Magnetosomes in Bacteria.

Berlin: Springer, 2007. 82. Print.

Schwermer, Carsten Ulrich, Timothy G. Ferdelman, Peter Stief, Armin Gieseke, Nastaran Rezakhani, Jaap

Van Rijn, Dirk De Beer, and Andreas Schramm. "Effect of Nitrate on Sulfur Transformations in

Sulfidogenic Sludge of a Marine Aquaculture Biofilter." FEMS Microbiology Ecology 72 (2010): 476-84.

Print.

Seitz, H.-J. & Cypionka, H. (1986). Chemolithotrophic growth of Desulfovibrio desulfuricans with

hydrogen coupled to ammonification of nitrate or nitrite. Arch Microbiol 146, 63–67.

Sievert, Stefan M., Ronald P. Kiene, and Heide N. Schulz-Vogt. "The Sulfur Cycle." Oceanography June

2007: 117-22. Web.

Skyring, G. W. (1987). Sulfate reduction in coastal ecosystems. Geomicrobiol. J. 5: 295-374 Teske, Andreas, Cathrin Wawer, Gerard Muyzer, and Niels B. Ramsing. "Distribution of Sulfate-Reducing

Bacteria in a Stratified Fjord (Mariager Fjord, Denmark) as Evaluated by Most-Probable-Number Counts

and Denaturing Gradient Gel Electrophoresis of PCR-Amplified Ribosomal DNA Fragments." Applied and


Teske, Andreas, Cathrin Wawer, Gerard Muyzer, and Niels B. Ramsing. "Distribution of Sulfate-Reducing

Bacteria in a Stratified Fjord (Mariager Fjord, Denmark) as Evaluated by Most-Probable-Number Counts

and Denaturing Gradient Gel Electrophoresis of PCR-Amplified Ribosomal DNA Fragments." Applied and


West, Nyree J., and David J. Scanlan. "Niche-Partitioning of Prochlorococcus Populations in a Stratified

Water Column in the Eastern North Atlantic Ocean." Applied and Environmental Microbiology 65.6

(1999): 2585-591. Print.

Wu, Xiu-Juan, Jian-Liang Pan, Xiang-Long Liu, Jing Tan, Dao-Tang Li, and Hong Yang. "Sulfate-reducing

Bacteria in Leachate-polluted Aquifers along the Shore of the East China Sea." NRC Research Press 55

(2009): 818-28. NRC Research Press. 10 July 2009. Web. 15 Dec. 2011.


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Figures

Figure 1. Map of Siders Pond and surrounding Falmouth area.

Figure 2. Baythmetry map of Siders Pond

Figure 3. Diagram of filtering rig set-up used for DNA Extraction

Figure 4. Physical profile of the examined Siders Pond water column.

Figure 4b. pH of water column up close

Figure 5. Chemical profile of the examined Siders Pond water column.

Figure 6. Cell abundance profile of the examined Siders Pond water column.

Figure 7. DAPI pics

Figure 8. filter colors

Figure 9. Cluster dendrogram of sequence similarity index

Figure 10. Venn Diagram

Figure 11. Phylogenetic Tree

Tables

Table 1. Volume of water filtered, color of filters, concentration of extracted DNA

Table 2. Detection of dsr at different depths

Table 3. Number of operational taxonomic units in sequences based on similarity cutoff

Table 4. Number of times each taxonomic unit was found in each level of the water column.

Sarah Nalven

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Figure 1. Map of Siders Pond (Google Maps) in Falmouth, MA. Note the channel that occasionally

connects Siders Pond to Martha’s Vineyard Sound.

Figure 2.Bathymetric map of Siders Pond (depths shown in meters). Star indicates 15 m deep hole;

water sampling took place close by at 12.2 m (graphic adapted from Giblin, 1990).


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Figure 3. Filtering set-up for DNA extraction.

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Figure 4. Profile of temperature, salinity, and dissolved oxygen in the Siders Pond water column.

0

2

4

6

8

10

12

0 5 10 15 20 25

De

pth

(m

)

Temperature (°C), Salinity (‰), Dissolved Oxygen (ppm)

Temperature

Salinity

Dissolved Oxygen


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Figure 5. Profile of pH in the Siders Pond water column.

0

2

4

6

8

10

12

14

6.7 6.8 6.9 7 7.1 7.2 7.3 7.4 7.5 7.6

De

pth

(m

) pH

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Figure 6. Profile of measured sulfide and sulfate values, as well as expected sulfate values (based on

salinity) in the Siders Pond water column.

0

2

4

6

8

10

12

0 2000 4000 6000 8000 10000 12000 14000 16000 18000

De

pth

(m

) Concentration (uM)

Measured Sulfate

Expected Sulfate

Sulfide


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Figure 7. Profile of cell abundance in the Siders Pond water column.

Figure 8. DAPI stained images from 2 meters and 10 meters depth.

0

2

4

6

8

10

12

5.E+06 1.E+07 2.E+07 2.E+07 3.E+07 3.E+07 4.E+07 4.E+07

de

pth

(m

) cells/mL

OXYCLINE

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Depth (m)

Volume Filtered (mL)

Filter Color Nucleic Acid Concentration (ng/uL)

2 960 light yellowish white 29.0

4 820 light yellowish white 19.9

4.5 830 pale lime green (purple water came out) 62.8

5 655 beige (purple water came out) 61.5

5.5 210 beige 27.9

6 620 dark brown 40.9

7 630 smoky gray 9.2

8 530 lighter beige 27.7

9 680 --- 39.3

10 615 lighter gray 23.5



Table 1. Volume of water filtered for each sample, color of filter, and concentration of nucleic acids

extracted from the water sample using these filters.

Figure 9. Different colored SterivexTM filters during DNA extraction.


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Depth (m)

dsr present? band description

2 maybe faint

4 maybe faint

4.5 yes bright

5 yes bright

5.5 yes bright

6 yes bright

7 yes bright

8 yes bright

9 yes bright

10 yes bright

11 yes bright

12 yes bright

Table 2. Detection of the dsr gene in the Siders Pond water column

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Cutoff (%) # OTUs

unique 106

1 37

2 26

3 25

4 22

5 21

6 19

8 17

Table 3. Number of operational taxonomic units in sequences based on similarity cutoff.

Sample 4.5 m 8 m 12 m

OTU 1 0 0 3

OTU 2 3 0 0

OTU 3 0 0 1

OTU 4 28 34 0

OTU 5 0 0 1

OTU 6 0 0 1

OTU 7 0 0 4

OTU 8 0 0 1

OTU 9 1 0 10

OTU 10 0 0 2

OTU 11 1 0 1

OTU 12 2 4 0

OTU 13 0 0 1

OTU 14 0 0 12

OTU 15 0 2 0

OTU 16 1 0 0

OTU 17 1 0 1

TOTAL 37 40 38

Table 4. Number of times each taxonomic unit was found in each level of the water column.


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Figure 10. Venn diagram depicting number of OTU sequences shared between 4.5, 8, and 12 m

communities. Circle size represents the approximate OTU sequences isolated from each depth.

Sarah Nalven

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Figure 11. A cluster dendrogram illustrating results from the Bray Curtis similarity index between OTU

sequences from 4.5, 8, and 12 m.


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Figure 12. Phylogenetic tree with sequences from Siders Pond and most similar dsr sequences\

Figure 13. A cluster of the phylogenetic three that contains sequences all isolated from 12 m and related

to a phenanthrine mineralizing sulfate-reducing bacterium.

Diversity and Distribution of sulfate-reducing …Many bacteria can use sulfur compounds in...

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