The microbiology of table salts

Seana P. Thompson

Cooperative Institute for Research in Environmental Sciences and Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado, USA

Honors Thesis Committee: Noah Fierera, Abby Hickcoxb, Ravinder Singhc, Alison Vigersd a Department of Ecology and Evolutionary Biology, Cooperative Institute for Research in Environmental Sciences, University of Colorado, Boulder, Colorado, USA b Arts and Sciences Honors Program, University of Colorado, Boulder, Colorado, USA c Department of Molecular, Cellular, Developmental Biology, University of Colorado, Boulder, Colorado, USA d Thesis Advisor. Department of Molecular, Cellular, Developmental Biology, Department of Psychology and Neuroscience, University of Colorado, Boulder, Colorado, USA

Defense Date: April 3rd, 2020

Abstract The dominant bacteria and archaea on table salts from around the world were identified and how these halophilic families varied as a function of salt chemical makeup was investigated. Through this study, the dominant taxa found on table salts were reported to be: Haloferacaceae, Pseudomonadaceae, Halomicrobiaceae, Halobacteriaceae, Nanohaloarchaeaceae, Rhodothermaceae, Burkholderiaceae, and Rhizobiaceae Marinobacteraceae, Alteromonadaceae, Rhodobacteraceae, Micrococcaceae, Moracellaceae, Staphylococcaceae, Oligoflexaceae, Halomonadaceae, and Flavobacteriaceae. The relative abundance of these microorganisms across samples was variable and related to the chemical composition of the table salts, including concentrations of iron (Fe), potassium (K), sodium (Na), magnesium (Mg), and calcium (Ca). The microbial families clustered into three general clusters. The three clusters were defined by Fe/K abundance, Na abundance, and Ca/Mg abundance. The Fe/K cluster showed the smallest range of microbial diversity and hosted a community dominated by Haloferacaceae. The Na cluster also hosted a dominant community of Haloferacaceae. This cluster also had a greater range of other microbes including Pseudomonadaceae, Halomicrobiaceae, Halobacteriaceae, Nanohaloarchaeaceae, Rhodothermaceae, Burkholderiaceae, and Rhizobiaceae. The Ca/Mg cluster showed the greatest diversity of microorganisms including Haloferacaceae, Pseudomonadaceae, Halomicrobiaceae, Halobacteriaceae, Nanohaloarchaeaceae, Rhodothermaceae, Burkholderiaceae, and Rhizobiaceae Marinobacteraceae, Alteromonadaceae, Rhodobacteraceae, Micrococcaceae, Moracellaceae, Staphylococcaceae, Oligoflexaceae, Halomonadaceae, and Flavobacteriaceae. These finding indicate a potential correlation between certain elements present in table salts and the microorganisms that reside there. Diverse microbial families thrive on table salts of varying composition, emphasizing the unique microbiology of a widely used food item.

Introduction

Environments with high concentrations of salt are considered extreme. These places foster microbial families that are unique from more hospitable environments because microorganisms must have a high salt tolerance to survive. Even something as small as a grain of salt can host a microbiome that is distinct from other environments. Microbes that thrive in salty conditions are called halophiles, and they are found across all three domains of life. Within the Bacteria domain of life, halophiles are found within the Fig. 1 The set of 40 table salt samples received from the Robert Dunn Lab at North Carolina State University. Cyanobacteria, Proteobacteria, Firmicutes, Photo taken by Lauren Nichols from the Robert Dunn Lab. Actinobacteria, Spirochaetes, and Bacteroidetes phyla [7]. Within the Archaea domain, halophilic microorganisms are found in the class Halobacteria and in Methods the order Methanococci [7]. Halophiles and non-halophilic microbes are often found A set of 40 table salt samples was collected within the same lineages. However, certain by the Robert Dunn Lab at North Carolina cohorts are made up entirely of halophilic State University from various salt species. These halophile-specific groups are companies across the world (Fig. 2). This set the order Halobacteriales of family was sent to the Fierer Lab and were stored in Halobacteriaceae (Euryarchaeota), the the -20C freezer to arrest microbial growth. order Halanaerobiales (Firmicutes) of The following procedures were followed for bacteria, the family Halomonadaceae the 40 samples examined in this study. (Gammaproteobacteria). Table salt is known generally by the chemical formula NaCl. However, there are Filtering Protocol other elements present in salt and each salt has a unique elemental makeup. With this in 0.25 grams of each salt was weighed and put mind, and the knowledge that different in individual 50 mL conical tubes. 50 mL elemental abundances can host unique microbial families, elemental composition of sterile water was measured into each tube. Tubes containing salt and sterile water were environments could be used to explain vortexed until the salt was fully dissolved. variation in microbial families. Specifically, The contents of each tube were poured over the elemental composition of different table a filter and filtered via vacuum filtration. salts could explain some of the apparent Another 50 mL of sterile water was poured variation in microorganisms across different salts.

Fig. 2 The metadata that was received with the collection of salt samples. Not all information was available for each sample.

An Invitrogen SequelPrep Normalization over the same filter and filtered via vacuum Plate (96) Kit was used to normalize the filtration. The filter was then removed with amplified PCR products. tweezers and put in individually labeled The microbial dataset was produced whirlpak bags and stored in a -20C freezer. using Illumina DNA sequencing, in which When original samples contained enough DNA is amplified, sequenced, and then salt to run the protocol again, duplicates analyzed. More information on sequencing were taken. can be found in the references [1]. Note: Filter blanks, extraction blanks, DNA Extractions and PCR blanks were all done as controls to compare to microorganisms found in all Salt sample filters were cut into pieces on samples. sterile petri dishes with tweezers and razor blades to make them the correct size to fit Salt Samples Chemical Analysis into DNA extraction single tubes. After the filters were cut and placed into DNA Each salt sample was chemically analyzed extraction single tubes, the DNeasy for presence and abundance of iron, PowerSoil Kits (Qiagen, DE) instructions potassium, magnesium, calcium, and sodium were followed. using inductively coupled plasma mass spectrometry. Mantel tests were run using PCR Bray-Curtis transformation to test for correlations between chemical elements. 16S rRNA gene PCR was performed on the extracted salt samples to amplify the DNA. Excel and R Studio Analysis Gel electrophoresis was done to verify the success of the PCR and DNA extractions Raw data was processed and filtered using using agarose gel and a DNA ladder for standard methods via DADA2 [1]. Blanks comparison. The gel was made with 135 mL were found to have very low community of Syber Safe and 2.5 grams of agarose. It abundance compared to the majority of the was run at 140 V for 45 minutes. samples. Thus, a cutoff of 8,000 total ASV reads was used to remove the blanks from downstream microbial analysis. Chloroplast, check if chemical elements were driving mitochondria, eukaryote, unassigned, and microbial community difference by NAs were filtered out and data was rarefied comparing individual matrices of chemistry to a depth of 8,000. Taxonomic relative and microbial data. PCoA was made to abundances were summarized. Using the extract ordination score and check metadata that came with the salt samples, correlation with chemical variables. PCoA dissimilarity matrices and ordination plots with Hellinger transformation was graphed were used to try to find clustering of the using Bray-Curtis Matrix and different samples. Ordinations were done with the metadata factors to find possible explanation metadata “Color”, “Type”, “Salt”, and for clusters. Chemistry data vectors were “Continent”. Microbial taxonomy was added to the microbial clustering graph. plotted in stacked bar plots at the order and Heatmaps for chemical abundance in family levels. Duplicates were dropped since samples and heatmaps for dominant duplicates were not able to be done for all microbial taxa in samples were created using samples. A taxa dissimilarity matrix was the ggplot2 package [2] and using Bray- calculated using the Bray-Curtis square root Curtis Square Root and Hellinger transformation. A mantel test was used to transformations.

Fig. 3 The top 50 ASV reads from the microbial sequencing data. A majority of the top ASVs are salt- specific microbes.

Fig. 4 Stacked bar plot showing the top 19 microbes found in the salt samples at the order level. “Na” is an unknown order of the Millicutes class of microbes.

Results range of variation in families. There was a total range of 4,709 unique ASVs. Sequencing shows dominant microbial Ordinations done with the metadata “Color”, taxa found to be salt-specific microbes “Type”, “Salt”, and “Continent” were not valuable for explaining the clustering of Sequencing of the DNA extracted from the microbial samples. Without different salt samples gave us taxonomy (taxa) metadata, only stacked bar plots comparing information about the microorganisms found the abundances of microbial families within on these samples. The top microbial ASVs each sample could be represented (Fig. 4). provided insight into the dominant taxa on table salts (Fig.3). Chemical analysis yields abundances of While these were the dominant taxa six different elements in samples found in the samples, there was still a great Fig. 5 Element abundances in the table salt samples vary widely. Na is the most abundant element across all samples except sample S034 (S_34). K and Ca have a wide range of abundances across samples while Fe is generally low across all samples except a few. Mg is only present in high amounts in sample S034 (S_34).

The abundances of iron, calcium, led to the hypothesis that perhaps chemical magnesium, potassium, and sodium were elements could be used to identify clusters measured in each sample by chemical in the microbial data. analysis (Fig. 5). None of the chemical elements showed a The chemical analysis showed almost all strong correlation with another element, samples contained high amount of sodium, except for Na and Mg, which had a which was as expected. Every sample except significant correlation reported at -0.91 (Fig. for one contained very little magnesium. 6). This shows an inverse relationship, Iron, potassium, and calcium abundances meaning as one element’s abundance is were varied across samples. The variance in high, the other element’s abundance is low. abundances of iron, potassium, and calcium sample size and perhaps these numbers could be improved in a larger study.

Fig. 6 Sodium (Na) and Magnesium (Mg) show a strong inverse relationship. None of the other elements show a strong relationship to each other.

This relationship is reasonable due to the extremely low abundance of Mg found in all samples except S_34, and the high abundance of Na found in all samples except S_34. These correlations helped make sense of the chemical data’s relationship to the microbial data in downstream analysis.

Chemical element abundance as a possible explanation for microbial families Fig. 7 (top) From a dissimilarity matrix calculated using the Bray-Curtis Square Root Transformation and a chemistry dissimilarity matrix calculated using the same Dissimilarity matrices of both microbial and method, the samples appeared to group into three different chemical data resulted in the microbial data clusters. Fig. 8 (bottom) Chemical element data clustering into three distinct groups (Fig.7). correlations to microbial data were run using individual matrices and mantel tests. Mg, Ca and Na are significant, Correlation tests between chemical but all have low correlation to the microbial data. Fe and abundance and microbial data provided p K are not significant and have very low correlation to the and R values (Fig.8). These tests microbial data. demonstrate that only Mg, Ca, and Na have Different metadata was compared to a significant relationship with microbial microbial data in an attempt to analyze the data, and all of these correlations are still cause of the clustering. Metadata “Color”, low. Fe and K did not have significant “Salt”, and “Continent” do not explain the relationships with microbial data, and their clustering of the microbial data. However, correlations to microbial data were very low. chemical element vectors did seem to However, this could be due to a small explain the clustering of the microbial data into three groups (Fig. 9). Fig. 9 PCoA/Hellinger and Bray-Curtis Square Root transformations with chemical data vectors added. This representation provides some explanation for the microbial samples clustering in three groups.

One cluster is explained by increased of the other top 20 microbes. In the Na abundance of Fe and K, another by cluster, almost every sample has a high increased abundance of Na, and the third by abundance of Na while no other element increased abundance of Ca and Mg. reaches a high abundance. The Na cluster shows Haloferacaceae to be the dominant Top microbial taxonomy explained by taxa in all samples; however, other microbes abundance of chemical elements show high abundances as well, suggesting that the Na cluster has a greater range of Patterns in specific microorganisms present microbial diversity than the Fe/K cluster. in the different clusters were visualized The Ca/Mg cluster shows a wide range of using heatmaps (Figs. 10). This elucidated elements with high abundance, specifically the relationships between chemical in this cluster Ca is generally in high abundance and certain taxa. abundance. Mg is only in high abundance in Based off these heatmaps, the Fe/K sample S_34 and Fe is in very low cluster had samples with high abundances of abundance throughout all samples in this K and/or Fe in common. Every sample in cluster. The Ca/Mg cluster shows a greater this cluster had a high abundance of Haloferacaceae and lacked high abundances Fig. 10 (Left side) Heat map of the abundances of individual elements in comparison to others within each sample. On the right side of the heat map, samples are organized into three clusters. The top-most cluster is defined by Fe/K, the middle by Na, and the bottom-most by Ca/Mg. (Right side) The abundances of microbial taxonomies in each sample relative to each other.

diversity of microbes than the other two abundances across samples while Fe and Mg clusters. This cluster showed high amounts remained relatively low in most samples. of Halomicrobiaceae as well as other Although correlation and significance microbes, although there is still generally a tests showed a weak relationship between high amount of Haloferacaceae throughout chemical abundance and microbial families, the cluster. Sample S_34, which is also the a pattern was still evident. Correlation tests only sample that shows almost no Na, and matrices showed that element showed distinct dominant taxa compared to abundance of salts seems to drive microbial the other samples. Three samples showed clustering to some extent. Perhaps the small high amounts of Pseudomonales, which are sample size of this study resulted in weaker bacteria found in a wide range of significance and correlation results. environments. The Fe/K cluster (Fig. 11) indicates that higher abundances of Fe/K might cause increased dominance of Haloferacaceae Discussion over other microbes. In this cluster, only sample S_38 contains a high amount of Fe As expected, the dominant microbial taxa on which indicates that K might be more the salt samples were microbes commonly responsible for the microbial families found in high-salinity environments. present in these samples. Specifically, It was also expected that the table salt increased levels of K seem to increase the samples have the highest abundance of Na dominance of Haloferacaceae. Halophilic over all other elements. NaCl, the commonly microorganisms are known to accumulate used chemical formula for table salt, KCl as a strategy to balance their cytoplasm indicate that Na is a key component in the [7]. Therefore, it is not unexpected that chemical makeup of salt. However, other increased levels of K would increase elements are also present in table salt, and dominance of a halophile. However, both Fe via chemical analysis we were able to detect and K were deemed not significant to and even trace amounts of the six elements that had very low correlation to the microbial were tested for. Ca and K had varying data. Fig. 11 (Top table) The Fe/K cluster shows near complete dominance of Haloferacaceae. In the sample (S_38) containing a high abundance of Fe, there was also a high abundance of the microbe, Halobacteriaceae. (Middle table) The Na cluster shows high abundance of Haloferacaceae.. It also shows increased abundances of other key microbes. This indicates that this cluster has a greater range of microbial diversity than the Fe/K cluster. (Bottom table) The Ca/Mg cluster has the greatest range of microbial taxa. This is in part due to sample S_34, which has the most unique microbial families out of all samples. (Key: from Heat Map numbers) Dominant Taxa means rating greater than 50, Moderate Abundance means rating in 50 to 12 range, Low Abundance means rating in 12 to 4 range.

The Na cluster (Fig. 11) samples other clusters. The samples in this cluster generally have high abundances of contain increased abundances of Haloferacaceae. This is an expected result Haloferacaceae, Pseudomonadaceae, given that Haloferacaceae thrive in Halomicrobiaceae, Halobacteriaceae, environments with high salinity and Na is a Nanohaloarchaeaceae, Rhodothermaceae, major component making up most table Burkholderiaceae, and Rhizobiaceae salts. However, this cluster, unlike the Fe/K Marinobacteraceae, Alteromonadaceae, cluster, also shows a greater diversity of Rhodobacteraceae, Micrococcaceae, other microbes. Haloferacaceae, Moracellaceae, Staphylococcaceae, Pseudomonadaceae, Halomicrobiaceae, Oligoflexaceae, Halomonadaceae, and Halobacteriaceae, Nanohaloarchaeaceae, Flavobacteriaceae. Some of this increase in Rhodothermaceae, Burkholderiaceae, and diversity is due to sample S_34, which is the Rhizobiaceae are also present in these only sample with a high abundance of Mg samples in increased abundance. and low abundance of Na. This sample In the Ca/Mg cluster (Fig. 11), high contains microbial families completely amounts of Ca seem to increase the unique from all other samples. This sample microbial diversity of the samples. contains high amounts of Micrococcaceae, Halomicrobiaceae are the dominant taxa in Moracellaceae, Staphylococcaceae, and this cluster, unlike in the other two clusters. Pseudomonadaceae. No other samples show While there are still high amounts of increased amounts of Micrococcaceae, Haloferacaceae, there are also high amounts Moracellaceae, or Staphylococcaceae. of a greater diversity of microbes than in the Excluding the unique taxa only present in the sample with high Mg, this cluster still environments that are difficult to survive in, has the greatest range of microbial taxa out they still host an expansive richness of life. of all the clusters. This may indicate that higher Ca abundance results in greater nutrient availability for a more diverse Conclusion microbial landscape, or that Ca abundance increases competitive abilities of a greater The sequenced results showed that there is a range of microbes. correlation between certain chemical Many of the dominant taxa found in these elements and the microbes present in clusters are halophilic microorganisms and samples. Microbial taxa clustered into three thus would be expected to be found on table different groups, each of which were salts. These microoraganisms include: described by different chemical elements. Haloferacaceae [8], Halomicrobiaceae, These results suggest that the elements Halobacteriaceae [26], present in salt do play a role in determining Nanohaloarchaeacea, Halomonadaceae which microbial families are present, and [20], Rhodothermaceae [5], and dominant. Even though table salt is mostly Alteromonadaceae [15]. made up of sodium and sodium drives Aquatic/marine and soil microorganisms families of salt-living microbes, the varying were also found to be dominant taxa, and presence of other elements affects the range these include: Flavobacteriaceae [21], of richness in microbial families and the Marinobacteraceae [24], Rhizobiaceae [22], strength of competition between microbes. and Rhodobacteraceae [16]. This is possible This presents a promising direction for because aquatic and soil environments future research. A larger salt sample size in contain some of the same chemical elements a following project could yield stronger as salt environments and thus could harbor correlation/significance values between certain sub-species of microorganisms suited microbial family clusters and chemical to these environments. concentrations. The chemical concentrations The presence of Burkholderiaceae [13], of a larger selection of elements could allow Oligoflexaceae [23], Pseudomonadaceae for more insight into how exactly chemical [25], Micrococcaceae [17], Moracellacea elements may drive microbial landscapes. [18], and Staphylococcaceae [19] in certain Culturing the salt samples in a variety of samples suggests that a greater richness of chemical conditions would identify which microorganisms can survive in high salt microorganisms can actually survive in environments, other than just explicit these conditions. Culturing would also allow halophiles. These microbial families have for testing the tolerances that microbes have sub-species that can survive in a vast for varying concentrations of sodium and diversity of environments, including ones other elements. high in salt. The presence of these microbial families shows that although salts are extreme

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