Impact of Microorganisms

TWiM #202: This Frass Doesn’t Stink

Podcast and Annotation Information
  • Annotation by Ava Kennard, Nicole Gulati, Ronak Chandiwala, and Rashad El-ghannam, and Maggie Schlarman
  • Podcast audio by TWiM: Listen to TWiM #202 Podcast
  • Podcast transcript by Otter.ai and edited by Laurel Thompson and Grace Helle: Access Podcast Transcripts
  • Papers Discussed:
    • Ceja-Navarro JA, Karaoz U, Bill M, Hao Z, White RA, Arellano A, Ramanculova L, Filley TR, Berry TD, Conrad ME, et al. 2019. Gut anatomical properties and microbial functional assembly promote lignocellulose deconstruction and colony subsistence of a wood-feeding beetle. Nature Microbiology. 4(5):864–875. doi: 10.1038/s41564-019-0384-y.
    • León-Zayas R, Roberts C, Vague M, Mellies JL. 2019. Draft Genome Sequences of Five Environmental Bacterial Isolates That Degrade Polyethylene Terephthalate Plastic. Irene, editor. Microbiology Resource Announcements. 8(25):e00237-19. doi: 10.1128/MRA.00237-19.

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 9:49 minutes

The Most Interesting Things (according to students)

  • Wood-feeding beetles eat up to 4.5 times their body weight every day, which is equivalent to us eating 500 pounds of food every day.
  • Microbial signals may be present in the frass and interpreted by the microbes.
  • Frass (beetle fecal material) is a signal to the plant, which makes them excrete chitinase. Chitinase attacks the shell of the beetle.

This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page.  A version of this paper is available on the UC Berkeley open access repository: https://escholarship.org/uc/item/1vq8d3rw

1.2. Main paper; discussion starts at 26:14 minutes

The Most Interesting Things (according to students)

  • More than 480 billion plastic bottles are made each year. PET plastic is used in many different ways but takes years to decompose. Our oceans are becoming collection points for these plastics. A vortex of water pulls in the plastic off the coast of California,  and now the plastic accumulated is the size of Texas. The debris can hurt marine life by eating them.
  • Looking at environmental components such as UV light access and their effects on microbial abilities; UV enhanced PET degradation on some microbes in the study, but maybe the trash in the ocean does not allow for enough UV light to reach the microbes in the ocean.
  • A whole consortia of bacteria may be the solution to solving the problem of plastic degradation.

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

Snippet Main
Vision and Change Topics
  • Microbial Ecology (V&C_E)
  • Structure and Function (V&C_SF)
  • Impact of Microorganisms (V&C_IM)
  • Information Flow and Genetics (V&C_IFG)
  • Metabolic Pathways (V&C_MP)
  • Impact of Microorganisms (V&C_IM)
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 12 (ASM_12): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 12 (ASM_12): Intrinsic factors, such as genotype, metabolism, and cell structures, impact the survival and growth of microbes.
  • Fundamental Statement 23 (ASM_23): The health of the environment and all organisms (microbes, plants, humans, other animals) are closely linked and interdependent, as described by the One Health paradigm.
  • 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
  • Identify the relationships among the beetle’s gut structure, physiology, metabolic processes, and the microbiome of the compartments.
  • Recall the plant biomolecule and its impact on beetles.
  • Identify the conclusions of the study.
 S L
  • Propose an experiment to test a hypothesis discussed in the podcast.
 S H
  • Recall the environmental issues associated with polyethylene terephthalate (PET).
  • Identify the workflow used by the researchers to isolate and characterize PET-degrading bacteria.
 M L
  • Defend the choice of sampling site.
  • Predict the metabolic features of other hypothetical PET degrading bacteria.
  • Propose factors that affect the ability to find pollutant-degrading bacteria based on the workflow and outcomes of this study.
 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

  • Metagenomic Libraries (15:55): This is a collection of DNA fragments, representing the genetic material of multiple organisms, that are cloned into a vector and introduced into a host cell for functional or sequence-based analysis. In the study, 30,000 unique proteins were found, in which 10% were related to breaking down lignin, xylan, and cellulose in the beetle.
  • Shotgun Metagenomic Sequencing (SMS) (15:55–17:20): This is a method that analyzes all the DNA in a sample to identify the present microbial species in beetle microbiomes.The authors studied and sequenced 16 metagenomic libraries, and found 30,000 unique proteins.
  • Nuclear Magnetic Resonance Spectroscopy (NMR) (20:57–21:40): This is a technique that utilizes the magnetic properties of atomic nuclei to study molecular structure and dynamics. It was used to find the different chemical makeups of the microbiomes in the study and further determine what metabolic processes were used in various compartments of the beetle gut.
  • Fluorescence Microscopy (20:57 –  21:39): This is a technique that uses a fluorescent tag to label, and therefore visualize, specific organisms or substances not visible to the naked eye. In the study, it was used to look at the transit time of wood by putting fluorescent balls in wood to see what it looks like when it comes out as a frass (beetle fecal material).

4.2. Main Paper

  • Enzyme Assay (26:14–29:20): This is a laboratory technique used to measure the activity of a specific enzyme, or the amount of a specific enzyme present in a sample.
  • Bacterial Culture (32:14–34:08): This is the process of growing bacteria in a controlled laboratory environment, typically using nutrient-rich media. This method is used to identify, study, and sometimes produce bacteria, playing a crucial role in diagnosing infections, research, and even industrial applications. The researchers set up bacterial cultures of microbes that broke down plastic and left them for 6 weeks to see if they would reduce the amount of PET granules.
  • Nuclear Magnetic Resonance Spectroscopy (NMR) (34:32–39:17): This is a technique that uses the magnetic properties of atomic nuclei to study molecular structure, identity, and other properties. It provides detailed information about the chemical environment, structure, and dynamics of molecules by examining how nuclei interact with an external magnetic field. Researchers used this to measure degradation of PET, by measuring the byproducts and biodegradation of the PET.
  • Genome Sequencing (34:32–39:17): This is a process that determines the entire genetic makeup of an organism or cell type, revealing the order of its DNA or RNA base pairs. Whole genome sequencing of microbial strains, in this study, was done using Illumina Hi-Seq instrumentation.
  • Kyoto Encyclopedia of Genes and Genomes (KEGG) (34:32–39:17) : This is a comprehensive database that is commonly used for investigating gene ontology.  It integrates information about genomes, pathways, diseases, drugs, and chemical substances. It’s a widely used resource for analyzing and interpreting biological data, particularly for understanding gene function and the flow of information and energy within cells and organisms. It was used to find lipase-like enzymes and enzymes that are part of metabolic and biosynthetic pathways.

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

5.1. Snippet Paper

  • Aerotolerance (9:49–15:17): The midgut is highly aerobic because it has a thin wall for oxygen to diffuse into. The anterior hindgut is anaerobic because it has a thick wall that prevents oxygen diffusion.
  • Metabolic Processes (13:50–16:00): The different sections of the gut in wood beetles have different microbiomes, and an important component of this is that the aerobic and anaerobic activity differs between sections.
  • Plant Response to Beetles (18:01–19:15): Plants release chitinase to degrade the beetle’s shell in response to frass (beetle feces) presence.
  • Fluorescent Markers (21:20–21:50): The authors fed the beetles fluorescent spheres and then analyzed the produced frass to determine the transit time of the spheres.

5.2. Main Paper

  • Plastic Pollution (26:14–29:00): Plastic can take centuries to degrade and it is building up in oceans, leading to chemical leaks and harming marine life.
  • Microbial Metabolism (29:05–29:50): The team employs concepts of microbial metabolism to study if microbes have acquired an ability to break down plastics.
  • Lipase Activity Assay (31:38–35:00): Of the 192 lipases they found, they tested to see if they could break down PET bottles using UV treated PET to help kickstart the degradation process. After 6 weeks, they measured the amount of granular PET to see if degradation had worked. Degradation was measured through NMR.
  • Generation Time (43:30–44:12): Microbes have a much more rapid generation time (time to double population) compared to humans, so they may evolve faster to digest new polymers that humans can’t.

6. Podcast Questions

  1. What type of bacteria are found in the section of the beetle gut that has a thicker wall? Why?
    1. Anaerobic bacteria because there is less oxygen diffusion.
    2. Aerobic bacteria because there is less oxygen diffusion.
    3. Anaerobic bacteria because there is more oxygen diffusion.
    4. Aerobic bacteria because there is more oxygen diffusion.
  2. The gut microbiome population is _________ in the different compartments of the beetle gut likely because _________
    1. Different; the gut microbiome bacteria are populated using small spiracle openings that are present in the beetle exoskeleton.
    2. The same; the digestive processes occur in a continuum that needs all the different species present in each compartment.
    3. Different; the digestive processes and structure varies so the bacteria that live and function in each compartment are different.
    4. The same; the gut microbiome bacteria are populated by the fungal and bacterial species that are present in the decaying wood.
  3. What were the key takeaways in the first snippet paper?
    1. There were found to be 30,000 unique proteins within the metabolomic libraries.
    2. The authors determined the transit time of frass from the beetle parent to offspring.
    3. There were different metabolic processes and microbes in each gut compartment
    4. Wood beetles have 6 different gut compartments and all serve different purposes
  4. What enzymes do plants produce in response to frass, and how does it affect the beetles?
    1. Chitinase, degrades the beetles shell
    2. Chitinase, disturbs the beetles microbiome
    3. Lipase, degrades the beetles shell
    4. Lipase, disturbs the beetles microbiome
  5. The podcasters noted that the young beetles eat frass and hypothesized that this would allow the gut microbiome to be populated. How could you test the hypothesis?
    1. Compare the gut microbiome of beetles that feed their young with frass to those that don’t.
    2. Compare the gut microbiome of beetles of different ages when fed frass or sterilized frass.
    3. Analyze the gut microbiome of beetles at different life stages when treated with antibiotics.
    4. Analyze the gut microbiome of beetles to identify the bacteria originating from decaying wood.
  6. The podcasters noted many issues with the accumulation of polyethylene terephthalate (PET) in the oceans, specifically in the Pacific Garbage Patch. What were they?
    1. Turtles mistake it for food.
    2. It leaches radioactivity.
    3. Animals get caught in it.
    4. It leaches pollutants.
    5. It blocks sunlight to algae.
  7. The researchers made a good choice to try to isolate PET degrading bacteria from the Galveston Bay area. What made this a good choice?
    1. The Galveston Bay Hurricane of 1900 populated the area with unusual species.
    2. It was near the campus of both research groups, so it was convenient for study.
    3. It is located near a heavy use oil industry and would have oil-contaminated soils.
    4. The Galveston Bay Estuary is known to have an abundance of rare species.
  8. What was the workflow discussed by the podcasters for identifying PET-degrading bacteria?  Start with 1 as step 1.
    1. ______ Treat PET with UV
    2. ______ Use NMR to detect PET degradation products
    3. ______ Collect soils
    4. ______ Identify genes and categorize by function
    5. ______ Identify fatty acid liberating bacteria
    6. ______ Culture on olive oil-rhodamine medium
    7. ______ Detect PET granule loss
    8. ______ Culture bacteria with UV-treated PET
    9. ______ Genome sequencing
  9. If you identified another bacterium that could degrade PET, you would expect it to have  ______ activity because _______.
    1. High lipase; other PET degraders have high lipase activity
    2. High lipidogenic; all PET degraders have only catabolic processes
    3. Low lipase; all PET degraders have only anabolic processes
    4. Low lipidogenic; other PET degraders have low lipase activity
  10. If you wanted to perform a similar study to isolate degraders of another pollutant, what factors could influence your ability to isolate a microbe with the ability to break down that pollutant? [pick all that apply]
    1. The length of time that a particular pollutant has been present in the environment.
    2. Conditions, like oxygen levels or exposure to UV light, can affect microbial abilities
    3. Other microbes that are present in the same environment, such as competitive species.
    4. The polluted area’s concentration and the pollutant’s physical and chemical properties.

7. Figure Reading Exercises

The following are two figure reading exercises, one from the snippet paper (Figure 6) and one from the main paper (Figure 1).

7.1. First Figure Reading Exercise

7.1.1. Learning Objectives

Students will be able to: 

  • Analyze the schematic diagram and text to identify which species are present and which biochemical processes occur in each gut compartment.
  • Argue for how the structure/anatomy of the gut supports the microbiome which in turn supports the biochemical processes occurring in each compartment.
  • Predict effects of structural alterations on the microbiome and processes.
Experimental Background (Ceja-Navarro et al., Figure 6)

Wood beetles can digest four times their body weight of decaying wood into simpler molecules such as glucose, pyruvate, lactate, acetate, xylose and benzoate.  These are excreted in their fecal material (frass) which is used to feed their offspring.  Wood is particularly difficult to digest because it contains lignins and cellulose. So how the beetle accomplishes this is important for understanding the species, but it also might yield valuable information for production of biofuels and other compounds from otherwise unusable lignin and cellulose. In this study, Ceja-Navarro et al. (2018) used complementary structural, metagenomic, and metabolomic studies to investigate how the Passalid beetles and their gut microbes sequentially break down woody biomass across their unique gut compartments.  Their combined results and conclusions are diagrammed in a schematic that shows how the beetle anatomy, biochemical process, and gut microbiome populations act in a coordinated way.  Here,  FG = foregut, MG = midgut (thinner wall), AHG = anterior hindgut (thicker wall), PHG = posterior hindgut (thicker wall), and TT = tracheal tubes. The Solid rectangles indicate processes and dashed line rectangles indicate molecules.

7.1.2. Questions

  1. Which of the following gut sections are primarily involved with the depolymerization of wood into glucose and xylose?
    1. AHG and FG
    2. MG and PHG
    3. TT and AHG
    4. FG and MG
  2. Match the color, process/conversion, and bacteria present to the digestive compartment in which they are present.
compartment color processes microbes
a. _____ Foregut Dark brown  = D Depolymerization = 1 Clostridiales = Co
b. _____ Midgut Green  = G Fermentation = 2 Clostridium = Cl
c. _____ Anterior hind gut Orange  = O Homoacetyogenesis = 3 Erysipelothrix = Er
d. _____ Posterior hind gut Tan  = T Methanogenesis = 4 Lactococcus = La
e. _____ Tracheal tubes Yellow  = Y Nitrogen fixation = 5 Methanobrevibacter = Me
Novophingobium = No
Parabacteroides = Pa
Turicibacter = Tu
  1. What structural feature of the gut would make you expect to find aerobic bacteria in the midgut and anaerobic bacteria in the anterior hind gut?
    1. Chitinous shell
    2. Processes present
    3. Gut wall thickness
    4. Circulation level
  2. Which of the following best explains why spatial organization of microbial metabolism benefits the host beetle?
    1. It decreases reliance on external enzymes
    2. It allows metabolic byproducts to accumulate
    3. It supports a cooperative use of resources
    4. It minimizes microbial activity across the gut
  3. You find a mutant beetle with a thinner gut wall throughout their digestive tract.  Which bacteria would you expect to populate the gut of this beetle?  How would this affect the beetle’s offspring?
    1. You would find both types of bacteria throughout the gut; they probably would grow poorly.
    2. You would find both types of bacteria throughout the gut; they probably would grow well.
    3. You would find anaerobic bacteria throughout the gut; they probably would grow faster.
    4. You would find aerobic bacteria throughout the gut; they probably would not grow well.

7.2. Second Figure Reading Exercise

7.2.1. Learning Objectives

Students will be able to:

  • Identify key features in a box plot that are important for this experiment.
  • Identify the relationship of an isolate to a consortium and identify examples of each.
  • Defend key experimental design choices, such as choice of medium.
  • Analyze the data and make conclusions about which cultures showed PET degradation.
  • Propose a reason why the researchers saw an unusual effect for the full consortium relative to the individual strains.
  • Propose a follow-up experiment to determine the specific metabolic pathways responsible for PET decomposition.
Experimental Background (Ceja-Navarro et al., Figure 6)

Polyethylene terephthalate (PET) is used to produce 480 billion bottles each year, but it takes years to decompose.  Due to this slow decomposition rate, it also poses a number of environmental hazards to aquatic wild-life including the leaching of pollutants, blocking sunlight to photosynthetic algae, and disrupting aquatic food webs. In an effort to identify ways to reduce PET decomposition time, León-Zayas et al. (2018) set about trying to isolate PET-degrading bacteria.  They first isolated soil bacteria from a hydrocarbon-rich environment and tested them for lipase activity.  Those that tested positive were then incubated individually (isolates) or in combinations that were found in the same soil sample (consortium) with UV-irradiated PET granules in a medium lacking any other carbon source (LCFBM).  The PET granules were weighed prior to incubation and after 6 weeks. The changes in PET granule weight are displayed as box plots.* In this figure, LCFBM = liquid carbon-free basal medium.* If you need help with box and whisker plots, these are helpful references: Visualizing the box and whisker plot; Box plot guide from Atlassian

A box plot showing change in weight of isolates.

Figure 1. “The full consortium, including all five strains, degrades PET to a greater extent than does any individual isolate or individual consortium after a 6-week incubation. Granular PET (Sigma-Aldrich, St. Louis, MO) was pretreated overnight with UV radiation (250 nm). Cultures of the five individual strains were grown overnight in LB broth and diluted to an optical density at 600 nm (OD600) of 1 to ensure that equal amounts of bacteria were added to each sample. Aliquots of each overnight culture were washed with phosphate-buffered saline (pH 7.4) twice, and equal volumes of bacteria were added to the appropriate 10-ml culture of LCFBM with 0.1 g total PET (starting OD600, 0.02). The samples were incubated at 30°C without shaking for 6 weeks. A negative control with UV-treated granular PET in medium without inoculation was kept under the same conditions. The granular PET weight loss from consortium 9 (least square means [LSM]9, 2.1 mg; standard deviation [SD], 0.05) treatment was statistically significantly greater than that with isolate 9.2 (P = 0.0002). The granular PET weight loss from the full consortium (FC) (LSMFC, 3.15 mg; SD, 0.07) treatment was statistically significantly greater than that for all isolates (P < 0.0001). Error bars indicate 1 SD.” (Leon-Zayas et al. 2018)

7.2.2. Questions

  1. Change in PET weight was quantified to generate these data, which are displayed by box plots. Match the box plot feature with its description. (1 = mean; 2 = median; 3 = outlier; 4 = interquartile range; 5 = full data range excluding outliers)
    1. ______ Box
    2. ______ Horizontal line in box
    3. ______ Whiskers
  2. Based on the box plot visualizations, which isolate has the largest variability among its individual sample values?
    1. 9.1
    2. 9.2
    3. 10
    4. 13.1
    5. 13.2
  3. What is the purpose of using carbon-free basal medium (CFBM) in the PET degradation experiment?
    1. To prevent bacterial contamination of the cultures
    2. To ensure PET is the only carbon source available
    3. To accelerate the degradation of PET granules
    4. To supply nutrients that enhance microbial growth
  4. What is the relationship of isolates to a consortium?  Which isolates are part of a consortium?
    1. A consortium is composed of strains of a single species; two Bacillus strains.
    2. A consortium contains multiple individual isolates: isolate 9.1 and isolate 9.2.
    3. A consortium is a group of anaerobic bacteria; Eryipelothrix and Clostridiales.
    4. A consortium contains all the mutant variants; lipase-degrading mutant strains.
  5. Which bacterial isolate from sample 9 shows a statistically different ability to degrade PET compared to consortium 9?
    1. 9.1
    2. 9.2
    3. 10
    4. 13.1
    5. 13.2
  6. The full microbial consortium (FC) showed a greater PET weight change than adding up all the individual strain changes. What might this indicate?
    1. It included genetically modified bacteria with enhanced enzymatic activity.
    2. PET is more easily degraded in the presence of oxygen from multiple strains.
    3. The strains likely interact synergistically, enhancing overall degradation activity.
    4. Each strain independently degrades PET using different mechanisms than others.
  7. Examine the box plots for consortium 9 and 13. What could you do to identify whether these are truly different?
    1. Perform statistical tests and look at the p value.
    2. Estimate the difference by inspecting the median lines.
    3. Check if the sample sizes are the same or different.
    4. They are different because the boxes do not overlap.
  8. How could you begin to determine the metabolic pathways responsible for PET degradation in the most effective strain?
    1. Measuring pH changes in the growth medium
    2. Performing a Gram stain on the bacterial cells
    3. Analyzing the genome for lipase-related genes
    4. Growing the strains in nutrient-rich broth

8. Paper Information and Licensing

8.1. Snippet paper

  • Ceja-Navarro JA, Karaoz U, Bill M, Hao Z, White RA, Arellano A, Ramanculova L, Filley TR, Berry TD, Conrad ME, et al. 2019. Gut anatomical properties and microbial functional assembly promote lignocellulose deconstruction and colony subsistence of a wood-feeding beetle. Nature Microbiology. 4(5):864–875. doi: 10.1038/s41564-019-0384-y.
  • This article is not licensed for Creative Commons use; see the article’s copyright information. Thus, the abstract and figures cannot be copied here. Please see the article at the journal’s web page.  A version of this paper is available on eScholarship: https://escholarship.org/uc/item/1vq8d3rw

8.2. Main paper

  • León-Zayas R, Roberts C, Vague M, Mellies JL. 2019. Draft Genome Sequences of Five Environmental Bacterial Isolates That Degrade Polyethylene Terephthalate Plastic. Irene, editor. Microbiology Resource Announcements. 8(25):e00237-19. doi: 10.1128/MRA.00237-19.
  • This article is licensed for Creative Commons use using CC BY 4.0, which allows re-use and adaptation with proper attribution and notation of any changes. See the article on the journal’s web page.

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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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