Information Flow and Genetics

TWiM #166: Dark Fermentation

Podcast and Annotation Information
  • Annotation by Leonardo Baumgartner, Martin Leyhe, and Triston Walsh, Nancy Boury, and Rebecca Seipelt-Thiemann
  • Podcast audio by TWiM: Listen to TWiM #166 Podcast
  • Podcast transcript by Sarah Morgan: Access TWiM #166 Transcript
  • Papers Discussed:
    • Zost SJ, Parkhouse K, Gumina ME, Kim K, Diaz Perez S, Wilson PC, Treanor JJ, Sant AJ, Cobey S, Hensley SE. 2017.  Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains. Proc Natl Acad Sci U S A. 114(47):12578-12583. doi: 10.1073/pnas.1712377114
    • Saidi R, Liebgott PP, Gannoun H, Ben Gaida L, Miladi B, Hamdi M, Bouallagui H, Auria R. 2018. Biohydrogen production from hyperthermophilic anaerobic digestion of fruit and vegetable wastes in seawater: Simplification of the culture medium of Thermotoga maritima. Waste Manag. 71:474-484. doi: 10.1016/j.wasman.2017.09.042

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 1:55 minutes

The Most Interesting Things (according to students) 

  • Small changes in an organism, or virus in this case, can have a big impact on their behavior.
  • Viral proteins can be grown in insect cells and this process has lower turnaround times than egg-derived vaccines!
  • A universal influenza vaccine is being researched based on conserved sequences

“H3N2 viruses continuously acquire mutations in the hemagglutinin (HA) glycoprotein that abrogate binding of human antibodies. During the 2014–2015 influenza season, clade 3C.2a H3N2 viruses possessing a new predicted glycosylation site in antigenic site B of HA emerged, and these viruses remain prevalent today. The 2016–2017 seasonal influenza vaccine was updated to include a clade 3C.2a H3N2 strain; however, the egg-adapted version of this viral strain lacks the new putative glycosylation site. Here, we biochemically demonstrate that the HA antigenic site B of circulating clade 3C.2a viruses is glycosylated. We show that antibodies elicited in ferrets and humans exposed to the egg-adapted 2016–2017 H3N2 vaccine strain poorly neutralize a glycosylated clade 3C.2a H3N2 virus. Importantly, antibodies elicited in ferrets infected with the current circulating H3N2 viral strain (that possesses the glycosylation site) and humans vaccinated with baculovirus-expressed H3 antigens (that possess the glycosylation site motif) were able to efficiently recognize a glycosylated clade 3C.2a H3N2 virus. We propose that differences in glycosylation between H3N2 egg-adapted vaccines and circulating strains likely contributed to reduced vaccine effectiveness during the 2016–2017 influenza season. Furthermore, our data suggest that influenza virus antigens prepared via systems not reliant on egg adaptations are more likely to elicit protective antibody responses that are not affected by glycosylation of antigenic site B of H3N2 HA.” (Zost et al. 2017)

1.2. Main paper; discussion starts at 26:10 minutes

The Most Interesting Things (according to students)

  • Simplifying a process can make it more effective and less expensive, thus making it applicable in industrial settings.
  • Seawater provides almost the total nutrient requirements for Thermotaga maritima in the bioreactor
  • 24% of Thermotaga maritima consists of archaeal genes

This article is not licensed for Creative Commons use; see article copyright information. Thus, the abstract cannot be copied here.

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

Snippet Main
Vision and Change Topics
  • Information Flow and Genetics (V&C_IFG)
  • Evolution (V&C_E)
  • Microbial Ecology (V&C_ME)
  • Metabolic Pathways (V&C_MP)
  • Impact of Microorganisms (V&C_IM)
ASM Fundamental Statements
  • Fundamental Statement 2 (ASM_2): The diversity of microbes has arisen because of processes that include horizontal gene transfer, mutation, reassortment, recombination, and natural selection in varying ecological niches favor the growth and survival of certain variants.
  • Fundamental Statement 19 (ASM_19): Non-cellular infectious agents, such as viruses, prions, viroids, and satellites, are dependent on host cell processes in order to replicate.
  • Fundamental Statement 22 (ASM_22): Most microbes interact with hosts in beneficial or neutral ways, with a minority having a detrimental impact on their host.
  • Fundamental Statement 12 (ASM_12): Bacteria and Archaea exhibit extensive metabolic diversity, including nitrogen fixation, methane production, and anoxygenic photosynthesis, many of which are unique to these two domains.
  • Fundamental Statement 13 (ASM_13): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 14 (ASM_14): Extrinsic factors, such as abiotic and biotic interactions in the environment, can impact survival and growth of microbes.
  • Fundamental Statement 26 (ASM_26): Humans leverage microbes and their products to address problems and improve quality of life.

3.  Potential Learning Objectives for the Podcast

The student will be able to: Paper1 Order2
  • Recall the major culture systems used to produce influenza vaccines.
  • Identify why the 2014–2016 influenza vaccine was less effective than other years.
 S L
  • Predict the effects of using different culture systems on vaccine efficacy.
 S H
  • Identify the features that are used to categorize Thermotoga maritima as an extremophile.
  • List some potential benefits of using bioderived hydrogen as a fuel source.
 M L
  • Design an experiment to determine what sea water components are necessary for Thermotoga maritima growth.
 M H

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

2 Learning 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

  • Culture Systems (see specific times for systems): These are systems for growing organisms.  Here the podcasters discuss a number of culture systems to grow virus or viral proteins including chicken eggs (10:50–11:30), HeLa cells (13:00–13:50), insect cells/baculovirus (17:30–19:00)  dog cells (19:45–20:00).
  • Vaccine Testing in Animal Models (14:50–15:00; 15:55–16:45): Different animals are used in biological research for different reasons.  Here, vaccine efficacy was tested using ferrets.

4.2. Main Paper

  • Culture Techniques (36:00–37:20): There are different culture needs for growing different organisms.  Their metabolic needs are determined by their ability to synthesize what they require for life.  Here, seawater was used as a substitute for an expensive mixture of nutrients to grow Thermotoga maritima.  This is an example of enriched media.

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

5.1. Snippet Paper

  • Glycoproteins (4:50–6:15; 9:30–10:30): Proteins can have sugars added to certain amino acids.  Hemagglutinin is a viral protein and a mutation resulted in a new glycosylation site that decreased vaccine efficacy.
  • Segmented Genomes (6:50–9:30): Influenza genome is segmented, making is unique among virus and highly variable over time.
  • Mutations and Reversion (11:30–12:00): When the flu virus is grown in chicken eggs, a mutation involving a glycosylation site on hemagglutinin was lost (reverted back to wild-type), limiting the vaccine effectiveness, (17:30-18:15) When grown in insects, the mutation in the glycosylated site in maintained.
  • Animal Model (14:50–15:00, 15:55–16:45): Ferrets are used as a model organism for influenza research.
  • Influenza Virus (18:30–19:00; 20:00–20:25; 21:00–21:45): Virus or viral proteins can be grown in a variety of living organisms for mass production.

5.2. Main Paper

  • Extremophiles (29:30): Thermotoga sp. are hyperthermophiles and live at 90C.  It was isolated from water near a volcanic island.
  • Horizontal Gene Transfer (32:00–33:30): This is when genetic material from another organism is integrated into another organism’s genome.  The researchers found that the Thermotoga sp. genome is 24% archaeal
  • Metabolic Diversity (31:35–31:55; 33:45–34:35): Thermotoga sp. carry out different metabolic processes than typical bacteria, with its catabolic activity producing H2 gas as a byproduct, (34:35; 40:30–41:46) H2 gas, the byproduct of Thermatoga sp. metabolic activity, has promise as a green fuel for the future. Its widespread use has largely been hindered by its high cost of production
  • Metabolic Needs (36:00–37:24): The growth medium for Thermotoga sp. must contain certain elements for growth.

6. Podcast Questions

  1. Which culture system is the most commonly used to produce the influenza vaccine?
    1. Chicken eggs
    2. Dog cells
    3. Ferrets
    4. HeLa cells
    5. Insect cells
  2. What was the major conclusion about what made the 2014–2015 vaccine less effective?
    1. A mutation occurred leading to reduced neuraminidase levels that affected viral assembly.
    2. A deletion occurred in the viral gene encoding hemagglutinin, an essential coat protein.
    3. A mutation occurred that led to antibody cross-reactivity which eliminated the viral proteins.
    4. A mutation occurred in the viral gene that led to a new hemagglutinin  glycosylation site.
  3. What was the major conclusion about why the revised 2015–2016 vaccine was still less effective?
    1. In the new, revised flu vaccine,  the neuraminidase structure was altered during injection.
    2. In the new, revised flu vaccine,  the egg-produced viruses reverted back to the non-mutant.
    3. In the new, revised flu vaccine,  the vaccine formulation triggered autoimmune suppression.
    4.  In the new, revised flu vaccine, the immune response targeted too many viral surface markers.
  4. If the vaccines for 2014–2015 and 2015–2016 had been produced mainly using the baculovirus system, what would have been the likely results?
    1. Ineffective for 2014–2015, but effective for 2015–2016
    2. Effective for 2014–2015, but ineffective for 2015–2016
    3. Ineffective for 2014–2015 and ineffective for 2015–2016
    4. Effective for 2014–2015 and effective for 2015–2016
  5. Which of the following are features of the extremophile Thermotoga maritima?
    1. Growth at high pressure.
    2. Growth at low oxygen.
    3. Growth at high temperature.
    4. Growth in high salt.
  6. Hydrogen is an excellent energy source, but still isn’t used much today. The researchers have identified a cost-effective way to produce hydrogen.  What are the benefits the podcasters discussed regarding their whole methodology?
    1. Their method converted hydrogen to non-flammable, so safety is increased.
    2. Their method used vegetable waste as substrate, so it reduces organic waste.
    3. Their method used sea water for many nutrients, so the cost is decreased.
    4. Their method used purified water as substrate, so it reduces energy activation.
  7. Which of the following is a reasonable first step for identifying what components of sea water are sufficient for Thermotoga maritima growth?
    1. Combine purified water and sea water in different ratios, grow Thermotoga maritima in each sample, look for T. maritima growth inhibition.
    2. Add different protease inhibitors to sea water, grow Thermotoga maritima in each mixture, look for T. maritima growth enhancement.
    3. Add different archaea to sea water and co-culture with Thermotoga maritima for each, look for when hydrogen production is present.
    4. Fractionate sea water in different ways, inoculate Thermotoga maritima in each fraction, look for T. maritima growth in each fraction.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 3) and one from the main paper (Figure 2A).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Identify key features of line-style neutralization plots.
  • Identify key experimental design features, such as controls and variable types.
  • Propose a control that would improve the experimental design.
  • Analyze the data to make conclusions about vaccination efficacy for each vaccine type.
  • Analyze the data to make conclusions about effect of the new glycosylation site on vaccination specificity.
Experimental Background (Zost et al., Figure 3)

The goal of vaccination is to have a long-lasting protection for those who are vaccinated.  While some have achieved this goal, such as for measles, other vaccinations have not, such as for influenza.  A major reason influenza vaccination is difficult is that these viruses have a high rate of mutation and genetic variability.  Influenza causes seasonal respiratory illness and the current methodology is to estimate which viral strains are likely to become prevalent and prepare a mixture vaccine against them.  The virus is a single-stranded RNA virus with a segmented genome that is packaged inside a lipid-protein particle that is spiked with primarily two proteins, hemagglutinin (HA) and neuraminidase (NA).  Following vaccination, antibodies are primarily formed against the HA protein.  In the 2014–2015 flu season, a variant with a HA mutation at codon 160 (K160T) became prevalent and caused vaccination to be ineffective.  This particular mutation introduced a new putative glycosylation site to the HA.  After finding this, the 2015–2016 mixture vaccine was updated to include this new variant, but vaccination effectiveness was variable.  Here, the researchers investigate the cause of this vaccination efficacy issue by examining antibody production and specificity.  They inoculated ferrets with the standard egg-grown vaccine strain that either is the wild-type version (K160; panel A) of hemagglutinin or has the mutation (T160; panel B) in hemagglutinin.  They assayed the ferret serum for antibodies that could neutralize each virus type (K160 or T160).  Recall that in neutralization assays, such as those used here to report the data, the lower dilution that is effective indicates the strongest binding.

line graphs
Figure 3. “Ferrets elicit different types of antibody responses when exposed to H3 viruses with K160 HA and T160 HA. Ferrets (n = 3 animals per group)were infected with viruses possessing (A) K160 HA or (B) T160 HA and sera were collected 28 d later. FRNTs were completed using viruses that possessedK160 HA or T160 HA. Neutralization titers are expressed as inverse dilution of sera that reduced foci by 90%. We completed three independent experiments with each sera. Shown are geometric means from the three in-dependent experiments. Statistical significance was determined using a paired Student’s t test.” (Zost et al. 2017)

 

7.1.2. Questions

  1. Which panel shows the data for the ferrets infected with the virus with the hemagglutinin that has a new putative glycosylation site?
    1. Panel A
    2. Panel B
  2. What is the most dilute serum used in this experiment?
    1. 1:1280 dilution
    2. 1:640 dilution
    3. 1:320 dilution
    4. 1:160 dilution
  3. The data for ferrets infected with virus K160 is shown in panel A. In this experiment _____ is the positive control.
    1. K160 antibody
    2. T160 antibody
    3. K160 virus
    4. T160 virus
  4. The data for ferrets infected with virus T160 is shown in panel B. In this experiment _____ is the positive control.
    1. K160 antibody
    2. T160 antibody
    3. K160 virus
    4. T160 virus
  5. In these experiments, there is no negative control. Which would have been good negative control(s) to include?
    1. Unvaccinated ferret serum
    2. Unvaccinated mouse serum
    3. A non-flu neutralization virus
    4. Glycosylation-free medium
  6. In which instances can you conclude that there are antibodies specific for only the EXACT codon 160 region of hemagglutinin used to infect the ferrets? What is your evidence?
    1. Ferrets infected with K160 HA virus; high dilution for panel A when challenged with K160 virus but not T160 virus.
    2. Ferrets infected with K160 HA virus; low dilution for panel A when challenged with T160 virus and with K160 virus.
    3. Ferrets infected with T160 HA virus; high dilution panel B when challenged with K160 virus and with T160 virus.
    4. Ferrets infected with T160 HA virus; low dilution panel B when challenged with T160 virus but not with K160 virus.
  7. What issue do you see regarding the positive control results when you compare these experiments?
    1. The T160 virus is not neutralized well by serum from ferrets immunized with T160 virus, so there must be something wrong with the T160 virus.
    2. The K160 virus is hyperneutralized  by serum from ferrets immunized with K160 virus, so there must be something wrong with the K160 virus.
    3. The T160 virus is hyperneutralized  by serum from ferrets immunized with T160 virus, so there must be something wrong with the K160 virus.
    4. The T160 and K160 viruses are neutralized equally by serum from ferrets immunized with T160 virus, so there is something wrong with the T160 virus.

7.2. Second Figure Reading Exercise 

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features of line diagrams related to these experiments.
  • Identify key features of the experimental design including controls and variable types.
  • Analyze the data to make conclusions about the impact of media type on compounds produced during fermentation.
  • Analyze the data to make conclusions regarding the suitability of natural sea water as a replacement for minerals in the fermentation.
Experimental Background (Saidi et al., Figure 2a)

There are international efforts to reduce dependence on fossil fuels for energy and one clean energy source that has not expanded in use is hydrogen.  This is likely because hydrogen is expensive to produce.  With this in mind, Saidi et al. (2017) began investigating a more cost-effective way to produce hydrogen, using fermentation with an extremophile species called Thermotoga maritima that naturally produces hydrogen.  Their further cost-cutting experiments involved testing whether natural sea water could provide nutrient salts and whether fruit and vegetable waste could contribute fermentable sugars. To test this, the researchers grew T. maritima in mineral basal medium (MBM; Experiment 1; E1) or natural sea water medium (NSM; Experiment 2; E2). Both fermentations contained model fruit and vegetable waste (MFVW). Lactate, and acetate liberated during the fermentation were quantified using high pressure liquid chromatography (HPLC) while hydrogen was quantified using gas chromatography (panel 2a).  Hydrogen productivity, which is a measure of the efficiency of hydrogen production, was also calculated and is shown as well (panel 2a).

This article is not licensed for Creative Commons use; see https://www.sciencedirect.com/science/article/pii/S0956053X17307110?via%3Dihub. Thus, the abstract and figures cannot be copied here.

7.2.2. Questions

  1. Which notation is for growth in natural sea water?
    1. E1; open symbols
    2. E2; filled symbols
    3. E1; filled symbols
    4. E2; open symbols
  2. The graph has one x-axis and two y-axes.  Which axes would you look at to find the amount of acetate liberated during fermentation? Which symbols would you need to find?
    1. Right y-axis; circles
    2. Left y-axis; diamonds
    3. Right y-axis; squares
    4. Left y-axis; triangles
  3. What statement best describes the conclusion about which medium is the best for acetate production?
    1. Acetate is produced most in MBM medium.
    2. Acetate is produced most in NSW medium.
    3. Acetate is produced well in both media.
    4. Acetate is not produced in either medium.
  4. Which medium allows hydrogen to be produced most efficiently in the shortest fermentation?
    1. MBM medium; hydrogen is produced most efficiently at 5 hours.
    2. NSW medium; hydrogen is produced most efficiently at 7.5 hours.
    3. Both media; hydrogen is produced equally efficiently at 22 hours.
    4. Neither media; hydrogen productivity is low in either medium.
  5. Based on these data, do sea water minerals support hydrogen generation?
    1. Yes, lactate and hydrogen are both produced in the sea water fermentation.
    2. No, acetate and hydrogen are not both produced in the sea water fermentation.
    3. Yes, acetate and hydrogen are both produced in the sea water fermentation.
    4. No, only acetate and lactate are produced in the sea water fermentation.

8. Paper Information and Licensing

8.1. Snippet paper

  • Zost SJ, Parkhouse K, Gumina ME, Kim K, Diaz Perez S, Wilson PC, Treanor JJ, Sant AJ, Cobey S, Hensley SE. 2017.  Contemporary H3N2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains. Proc Natl Acad Sci U S A. 114(47):12578-12583. doi: 10.1073/pnas.1712377114
  • This article is licensed for Creative Commons use using CC BY-NC-ND 4.0, which allows re-use for non-commercial purposes with proper attribution, but not derivatives. See https://www.pnas.org/doi/abs/10.1073/pnas.1712377114

8.2. Main paper

License

Icon for the Creative Commons Attribution 4.0 International License

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.

Share This Book