Microbial Ecology

TWiM #224: One Hundred Million Year Old Bacteria

Podcast and Annotation Information

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 03:45 minutes

The Most Interesting Things (according to students)

  • We found the concept that there are still live microbes in these scrolls that are thousands of years old very interesting. It is fascinating how these microbes, although some are spore-formers, were able to withstand the years and all the possible environmental changes that came with it. It also brings into question the methods of creation and preservation of these scrolls by ancient peoples, as well as if there are any unknown, pathogenic microbes present that are waiting to be discovered.
  • We also thought that the methods used to sequence and identify these microbes, specifically the fact that researchers were only able to use mere crumbs of the scrolls, were extremely interesting. It is already a daunting task to extract DNA and sequence it, but to do so from such small amounts of sample without introducing contamination is very impressive. Finally, another interesting concept was seeing how interdisciplinary the sciences are. In this paper, genetic analysis is used as a key method in the anthropology of the Dead Sea Scrolls. Also, science and religion are shown to enhance rather than discredit each other.

Abstract

The abstract cannot be copied due to licensing restrictions. Please see licensing information and links to the article at the journal’s web page and/or PubMed in Section 8.2.

1.2. Main paper; discussion starts at 17:04 minutes

The Most Interesting Things (according to students)

  • What we thought was most interesting about the main paper was seeing the great extent of conditions in which microbes can survive. These microbes, discovered from solid rock beneath the sea, lived in pores smaller than what’s required for movement. This means that these microbes were essentially living fossils. We’re only just beginning to understand the diversity of life and the amazing capabilities of microorganisms.
  • We found the wide range of microbial diversity listed in the main paper also fascinating. I had a basic idea that microbes could survive in extreme environments, but the wide range discovered in these environments were fascinating to me.
  • Finally, we found the lifespan of the bacteria mentioned in the main paper extremely interesting. From our general knowledge of microbiology, we knew that most bacteria could survive in ideal environments for at least a few years, but we had no idea that some could survive for 101.5 million years.

Abstract

“Sparse microbial populations persist from seafloor to basement in the slowly accumulating oxic sediment of the oligotrophic South Pacific Gyre (SPG). The physiological status of these communities, including their substrate metabolism, is previously unconstrained. Here we show that diverse aerobic members of communities in SPG sediments (4.3‒101.5 Ma) are capable of readily incorporating carbon and nitrogen substrates and dividing. Most of the 6986 individual cells analyzed with nanometer-scale secondary ion mass spectrometry (NanoSIMS) actively incorporated isotope-labeled substrates. Many cells responded rapidly to incubation conditions, increasing total numbers by 4 orders of magnitude and taking up labeled carbon and nitrogen within 68 days after incubation. The response was generally faster (on average, 3.09 times) for nitrogen incorporation than for carbon incorporation. In contrast, anaerobic microbes were only minimally revived from this oxic sediment. Our results suggest that microbial communities widely distributed in organic-poor abyssal sediment consist mainly of aerobes that retain their metabolic potential under extremely low-energy conditions for up to 101.5 Ma” (Morono et al. 2020)

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

 Snippet Main

Vision and Change Topics
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)
  • Metabolic Pathways (V&C_MP)
  • Microbial Ecology (V&C_ME)
  • Structure and Function (V&C_SF)

ASM Fundamental Statements
  • Fundamental Statement 3 (ASM_3): The evolution of microbes is impacted by their interactions with the environment and a variety of ecological forces, including other microbes, humans, and habitats.
  • Fundamental Statement 5 (ASM_5): The structure and function of microbes are revealed by the use of microscopy, culture, and metabolic analysis, molecular methods, and bioinformatic tools.
  • Fundamental Statement 20 (ASM_20): Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.
  • Fundamental Statement 8 (ASM_8): Microbes have unique genomes, structures, and/or biochemical characteristics that distinguish them from each other.
  • Fundamental Statement 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 20 (ASM_20). Microbes are ubiquitous, found in diverse and dynamic ecosystems, where they use available resources and often form complex communities.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recognize the technique used to identify the animals whose skins were used to make the Dead Sea Scrolls.

S

L
  • Speculate how the animal skins used to make the Dead Sea Scrolls contributes to the history of compiling the story.

S

H
  • Recall the features of the microbes living in the sea floor oxic sediments.

M

L
  • Defend the use of proper aseptic technique by determining how improper aseptic technique might affect these results.

M

H

1 Papers: Snippet (S) or Main (M)

2Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

  • Next-generation Sequencing (7:29–8:18; 14:09–16:25): Next-generation sequencing is a method of genetic analysis that is fast and able to analyze and give results for large sequence numbers. This provides nucleotide sequences that can then be further analyzed for specific regions or protein production.
  • DNA Fingerprinting (8:12–10:02; 13:19–14:09): DNA fingerprinting is a technique of microbe differentiation based on unique patterns found in their DNA, creating a “fingerprint” that can be used for identification.

4.2. Main Paper

  • Nanometer-scale Secondary Ion Mass Spectrometry (Nano SIM) (23:34–34:37): Nano SIM involves measuring specific elements and/or isotopic material in the test samples. This allowed the researchers to determine if carbon 13 or nitrogen 15 was incorporated into microbial biomass.
  • Redfield Ratios (23:34–34:37): This is the atomic ratio of carbon, nitrogen and phosphorus that is present in phytoplankton in the ocean. It was named after Alfred Redfield who discovered that the ratio of these elements in the ocean were 106 parts carbon, sixteen parts nitrogen and one part phosphorus.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

  • Genomics (7:29–10:16, 13:19–16:25): Genomics involves using genetics (DNA, RNA sequences) to determine information about the microorganism. This can include determination of the microorganism’s function, identification and ideal growth conditions. It’s a broad field and includes phylogenetics, next-gen sequencing, and DNA Fingerprinting.
  • Phylogenetics (13:19–14:09): Phylogenetics involves using DNA sequences of different bacteria and comparing them to see the similarities and differences. These similarities and differences are used to construct a phylogenetic tree which will reveal which bacterial species are related and what the last universal common ancestor (LUCA) of all the bacterial species is.

5.2. Main Paper

  • Anaerobic vs Aerobic Respiration (23:34; 36:03–38:27; 38:27):  Anaerobic respiration can’t occur using oxygen and requires the use of another terminal electron acceptor.  The microbes stopped growing without oxygen.  There was a discussion of  the importance of electron acceptors and what’s needed for microbes to live.
  • Binary Fission and Generation Time (35:15): These are both described with E.coli as an example. E.coli has a generation time of 20 minutes; the increase in growth on a logarithmic scale mentioned here indicates that bacteria reproduce by binary fission (one cell to two cells, two cells to four cells, etc.)

6. Podcast Questions

  1. Which techniques were used to identify the animals whose skins were used for the Dead Sea Scrolls? [pick all that apply]
    1. Polymerase chain reaction (PCR)
    2. Next generation sequencing
    3. Colony forming units on plates
    4. Plasmid DNA isolation
  1. Genetic analysis shows that the first half of the story of Jeremiah was written on cow skin, but the second half was written on lambskin. What additional information would infer that the story of Jeremiah was compiled from writings by different peoples?
    1. The cow and lamb skins are found to be indigenous to different regions.
    2. The cow and lamb skin writings comprise the two halves of the story.
    3. The cow and lamb skins are found to be indigenous to the same region.
    4. Cows were not introduced to this part of the world until after sheep.
  1. What were the features of the microbes growing in the oxic sediments? [pick all that apply]
    1. They used oxygen as an electron acceptor.
    2. They used fermentation to generate ATP.
    3. They grew very slowly compared to E. coli.
    4. They used only carbon 13 as their carbon source.
    5. They used nitrate as an electron acceptor.
  1. Proper aseptic technique is important in most microbial studies, but particularly in this one (101.5 million-year-old microbes).  If proper aseptic technique was not followed then ____________
    1. living microbes could have been introduced to the sample by contamination
    2. the microbes could have been killed during the experimentation process
    3. the sediment core could have picked up nearby nitrates and phosphorus
    4. the methodology for determining metabolic activity would have been skewed

7. Figure Reading Exercises

The following are two figure reading exercises, both from the main paper (Figures 1A-C & E-G and 2).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to:

  • Identify key features in fluorescent microscopy images overlaid with nutrient heatmaps.
  • Evaluate the data and make conclusions about what the microscopy image-heatmaps indicate about nutrient uptake and bacterial metabolism.
  • Argue what a fluorescence microscopy image communicates about microbial growth and metabolism
Experimental Background (Morono et al., Figure 1, A-C; E-G)

Sea sediments account for 70% of the Earth’s surface with its microbes contributing an estimated 12-45% of microbial biomass.  Little is known about the microbes that contribute such a large proportion of the Earth’s biomass because they are so difficult to sample.  An ocean drilling program called the Integrated Ocean Drilling Program Expedition (IODP) has allowed some preliminary examination of this habitat and its microbes.  Here, Morono et al. (2020) study the microbes taken from cores at the South Pacific Gyre.  With such extreme conditions, these researchers were interested in which types of metabolic processes, if any, were active in bacteria from the deep sea sedimentary core. To do this, the researchers incubated microbes from the oldest sediment (U1365 9H-3) with either 1) bicarbonate labeled with a carbon-13 isotope (13C) and ammonium labeled with nitrogen-15 isotope (15N) or 2) amino acids labeled with both 13C and 15N to track the biological activity of the cells.  Isotope tracing can be used to trace carbon and nitrogen exchange and circulation through an ecosystem, respectively. Since these isotopes are stable but heavier than their more abundant naturally occurring isotopes (12C and 14N), they can be successfully integrated into biomass and used in metabolic processes. Here, the position of representative sediment microbes is shown using a fluorescent DNA dye called SYBR green (panels a and e).  Cells were incubated with either 13C-bicarbonate and 15N-ammonium (panel a-d) or a 13C,15N-amino acid mix (panels e-h).  To quantify metabolism, several chemical ratios are shown as heatmaps (panels b, c, f, g; note the color scale bar at right of each image).  The ratio of13C/12C is shown as a heat map in panels b and f.  The ratio of 12C15N/12C14N [for simplicity, think of this as 15N/14N] is shown as a heat map in panels c and g.

A set of 8 microscopic images, with different luminescent effects shown in a, b, c, e, f and g. D an dh are greyscale.
Figure 1. “13C and 15N incorporation in representative microbial cells. Cells from incubations of U1365 9H-3 with 13C-bicarbonate and 15N ammonium (a–d) and 13C,15N-Amino acid mix (e–h). (a, e) SYBR Green I-stained cells under fluorescence microscopy. b, c, f, g Ratio images of 13C/12C (b, f) and 12C15N/12C14N ratios (c, g) of the same regions imaged in a, e, demonstrating locations of 13C and 15N incorporation. Color-scale ranges of the ratios are shown as numbers appearing at top and bottom of the color bar. The background membrane region, which is identified by fluorescence images, is excluded from the ratio calculation and shown as black background. d, h. Secondary electron (NanoSIMS) images of the same regions in a, e. Bars represent 5 μm. Similar images were processed for obtaining the dataset (Supplementary Data 1) of substrate incorporations for 6986 individual cells.” (Morono et al. 2020, no changes)

7.1.2. Questions

  1. Which panel shows the cells stained with SYBR green and what is the purpose of showing this?
    1. Panel a only; it shows us the size and shape of the microbes.
    2. Panels a and e; it tells us where the cells are located in the image.
    3. Panel d and h; it shows us where DNA replication is occurring.
    4. Panel a and d; it allows us to compare the cytoplasmic volume.
  2. Which scenario below indicates nitrogen uptake after incubating cells with ammonium labeled with 15N (nitrogen-15), and which heat map color would indicate this high nitrogen uptake?
    1. A high ratio of 15N to 14N; yellow
    2. A low ratio of 15N to 14N; blue
    3. A higher abundance of cells; green
    4. A lower abundance of cells; gray
  3. Which scenario below indicates that no carbon uptake occurred after incubating cells with bicarbonate labeled with 13C (carbon-13), and which heat map color would indicate this low carbon uptake?
    1. A higher abundance of cells; green
    2. A lower abundance of cells; gray
    3. A high ratio of 13C to 12C; yellow
    4. A low ratio of 13C to 12C; blue
  4. Which panels show the data for amino acid uptake after incubating cells with amino acids labeled with 15N and 13C isotopes?
    1. A ratio of 13C/12C (panel b); a ratio of 15N/14N (panel c)
    2. A ratio of 13C/12C (panel f); a ratio of 15N/14N (panel g)
    3. A ratio of 13C/12C (panel b); a ratio of 15N/14N (panel f)
    4. A ratio of 13C/12C (panel f); a ratio of 15N/14N (panel c)
  5. What does the amino acid uptake indicate about the cellular metabolism? What is your evidence?
    1. Only carbon metabolism is present; High ratio of 13C/12C (panel f, orange) and low 15N/14N (panel c; blue).
    2. Only nitrogen metabolism is present; Low ratio of 13C/12C (panel b, blue) and high 15N/14N (panel f; orange).
    3. Metabolism is not present; Low ratios of 13C/12C (panel b) and 15N/14N (panel c) as indicated by blue.
    4. Metabolism is present; High ratios of 13C/12C (panel f) and 15N/14N (panel g) as indicated by yellow.
  6. What do the combined data for bicarbonate, ammonia, and amino acid uptake indicate about the cellular metabolism? What is your evidence?
    1. The cells appear to utilize ammonia better than bicarbonate or amino acids; the ammonia uptake is higher (panel c) compared ammonia or amino acids (panels b, f, g).
    2. The cells appear to utilize amino acids better than bicarbonate or ammonia; the amino acid uptake is higher (panels f and g) compared bicarbonate/ammonia (panels b and c).
    3. The cells appear to utilize bicarbonate better than ammonia or amino acids; the bicarbonate uptake is higher (panel b) compared ammonia or amino acids (panels c, f, g).
    4. The cells appear to utilize bicarbonate and ammonia better than amino acids; the bicarbonate (panel b) and ammonia (panel c) uptake are higher compared amino acids (panels f and g).

7.2. Figure Reading Exercise 2

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of line and violin plots.
  • Analyze the data and make conclusions about microbial nutrient preferences for each set of sediment core microbes.
  • Defend the use of violin plots over bar plots for the represented data.
Experimental Background (Morono et al., Figure 2)

Sea sediments account for 70% of the Earth’s surface with its microbes contributing an estimated 12-45% of microbial biomass.  Little is known about the microbes that contribute such a large proportion of the Earth’s biomass because they are so difficult to sample.  An ocean drilling program called the Integrated Ocean Drilling Program Expedition (IODP) has allowed some preliminary examination of this habitat and its microbes.  Here, Morono et al (2020) study the microbes taken from cores at the South Pacific Gyre.  With such extreme conditions, these researchers were interested establishing the conditions which would support microbial growth. First, they quantified how nutrient supplementation affected microbial cell counts by incubating microbes from four different sediment cores ranging from 4.3 to 101.5 million years ago (Ma) in media supplemented with a variety of nutrients: bicarbonate, acetate, glucose, amino acid mixture, pyruvate, or ammonia. They quantified cell number over time (panel a; days 0, 21, 68, 557). They were next interested in how the nutritional supplements affected metabolism, so they quantified carbon and nitrogen incorporation using a nano-scale mass spectrometry technique called NanoSIMS (panels b and c).  These data are plotted as violin plots.  For additional information on violin plots, you can see https://www.atlassian.com/data/charts/violin-plot-complete-guide.

Plots showing carbon and nitrogen incorporation.
Figure 2. “Microbial responses to addition of carbon and nitrogen substrates. Plots for samples used for the incubations are aligned vertically. a Plots of cell abundance during incubation. Cell abundances for Incubation time 0 are abundances in the sediment samples before incubation was initiated. For the samples incubated with carbon substrates (bicarbonate, acetate, glucose, and pyruvate), ammonia was added as nitrogen source. The incubation labeled “Ammonia” received ammonia as the nitrogen source with no additional carbon. n = 76 samples (one for Incubation time 0 and three for Incubation sampling points [days 21, 68, and 557] per substrate for each sediment sample). b, c. Incorporation of carbon- (b) and nitrogen- (c) substrates by microbes identified by NanoSIMS cellular ROIs (Supplementary Data 1). Substrate incorporation for each ROI was plotted over kernel density violin plots. (Morono et al. 2020, no changes)

7.2.2. Questions

  1. Which color/shape line shows the cell abundance data when acetate is added to the growth medium?
    1. red circle
    2. blue triangle
    3. green square
    4. blue star
  2. At what number of days were the measurements done for cell counts using different supplements (panel a)?
    1. 0 days, 21 days, 68 days
    2. 0 days, 68 days, 557 days
    3. 21 days, 68 days, 557 days
    4. 0 days, 21 days, 68 days, 557 days
  3. Which sediment core microbe samples show an increase in cell number from day 0 to day 21 when glucose is added to the medium? [pick all that apply]
    1. U1365 8H-2
    2. U1365 9H-3
    3. U1365 2H-5
    4. U1365 1H-2
  4. For sediment core microbe sample U1365 8H2 (95.4 Ma), which nutrient supplement is initially supportive, but is not able to support microbial survival later? What is your evidence?
    1. bicarbonate; a steep drop between days 68-557
    2. acetate; a decrease in slope at day 68 to 557
    3. glucose; no increase in counts after day 68
    4. amino acid mix; a flat line slope until day 557
  5. Based on the media supplement preferences for each sediment core sample, are the microbes present likely the same or different? What is your evidence?
    1. Different; they are separated by millions of years.
    2. Same; they all increase by day 68 and remain active.
    3. Different; they have different growth requirements.
    4. Same; they all use pyruvate supplementation similarly.
  6. What does each color represent in the carbon and nitrogen incorporation data panels (b and c)?
    1. Different sediment samples
    2. Different species present
    3. Different types of substrate
    4. Different incubation time
  7. At what number of days were the measurements done for carbon and nitrogen incorporation using different supplements (panels b and c)?
    1. 0 days, 21 days, 68 days, 557 days
    2. 21 days, 68 days, 557 days
    3. 0 days, 21 days, 68 days
    4. 0 days, 68 days, 557 days
  8. For a violin plot, what would a dataset with a high value, such as high carbon incorporation (panel b) and little variation among the individual samples look like?  What is an example of this pattern in the data panels?
    1. a small shape with intense color high on the y-axis; 9H-3 pyruvate at day 557.
    2. a large shape with diffuse color all along the y-axis; 1H-2 acetate at day 557.
    3. a small shape with intense color low on the y-axis; 9H-3 bicarbonate at day 557.
    4. a large shape with intense color all along the y-axis; 2H-5 acetate at day 21.
  9. Which sediment sample core microbes are best at using/incorporating most, if not all, of the nutrient supplements?  What is your evidence?
    1. U1365 8H-2
    2. U1365 9H-3
    3. U1365 2H-5
    4. U1365 1H-2
  10. If these data (panels b and c) had been represented by bar plots rather than violin plots, what would be less clear about the data?
    1. The distribution of the data would be unclear.
    2. The mean of the data would be unclear.
    3. The sample type would not be clear.
    4. The type of nutrient used would not be clear.

8. Paper Information and Licensing

8.1. Snippet paper

  • Anava S, Neuhof M, Gingold H, Sagy O, Munters A, Svensson EM, Afshinnekoo E, Danko D, Foox J, Shor P, Riestra B, Huchon D, Mason CE, Mizrahi N, Jakobsson M, Rechavi O. 2020. Illuminating Genetic Mysteries of the Dead Sea Scrolls. Cell, 181(6), 1218–1231.e27. https://doi.org/10.1016/j.cell.2020.04.046
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here.

8.2. Main paper

License

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Podcast Annotation and Resources in Microbiology Copyright © 2025 by Rebecca Seipelt-Thiemann; Nancy Boury; Gwendowlyn S. Knapp; Amaya Garcia Costas; and Patrick Armstrong is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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