Bi 1x Spring 2021 Kombucha population genetics
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Bi 1x Spring 2021
Kombucha population genetics
1 Overview
In this lab, you will investigate the bacterial diversity in the microbial community
responsible for fermenting tea into tasty kombucha. Together, we will prepare tea-
based growth media for kombucha cultures and allow them to ferment for about 10
days. We will then survey the bacterial diversity in the developed cultures both by
taste (though see the important note about tasting kombucha below) and molecularly
by high-throughput sequencing of the 16S rRNA gene. We will perform state-of-the-
art analysis of the data using the QIIME2 package with cloud computing, bringing
the insights that modern sequencing and computational techniques enable to an an-
cient fermented beverage tradition. Along the way, you will learn to appreciate the
diversity of microorganisms around us and their enormous metabolic capacity. You
will also get an introduction to tools microbial ecologists use to study the structure
and function of complex microbial communities in nature.
2 Background
2.1 Microbes and the environment
Microorganisms, and specifically Bacteria and Archaea, are very diverse and en-
compass significant functional (metabolic) potential. The presence of these microor-
ganisms is often determined by the biotic and abiotic conditions in which they are
present. The Dutch microbiologist Lourens Gerhard Marinus Baas Becking (1895-
1963) described it best, “Alles is overal: maar het milieu selecteert,” or, in English,
“Everything is everywhere, but the environment selects.” A similar notion was posited
earlier by another Dutch microbiologist, Martinus Willem Beijerinck (1851-1931).
What does Baas Becking mean by his famous statement? He means that microbes
of vast variety are present in most ecosystems, but they are present in small numbers
and are inactive. They are therefore hidden from us, unless we look carefully. In each
environment, some microbes that are well-suited for that particular set of conditions
thrive. These are the ones we can most directly observe.
2.2 Microbial lifestyle
In order to study which organisms will thrive in which conditions, microbial ecol-
ogists and environmental microbiologists meticulously study the metabolism of mi-
croorganisms, investigating how the microbes convert carbon, nitrogen, phospho-
rus, and sulfur, to bioactive molecules. One approach is to study the metabolic dy-
namics of mesocosms, which are controlled samples of a local environment. Alter-
natively, we can sample our ecosystem of interest and bring it back to the lab to con-
struct a mini-ecosystem where we control and perturb different conditions in which
1
this ecosystem is maintained. We could, for example, control temperature, nutrient
availability, and pH. By controlling specific conditions, we can gain insights about
the lifestyle of the microbes in the mini-ecosystem we construct.
Microorganisms can utilize a different sources of energy and various carbon sources
that define its lifestyle. Those microorganisms that get energy from sunlight are pho-
totrophs, and those that get their energy from chemical compounds are chemotrophs.
Those that get electrons from organic compounds are organotrophs, and those that
oxidize inorganic compounds are lithotrophs. Organisms that rely on an organic car-
bon source are heterotrophs, while those that can fix carbon dioxide are autotrophs.
This nomenclature is in-fact made of all three groups of tropism: energy source-
oxidizing donor-carbon source. For example, cyanobacteria (or blue-green algae) are
photolithoautotrophs. Our goal in this experiment is to construct mini-ecosystems.
Hopefully, we will be able to observe various microbial lifestyles.
2.3 Kombucha
In this experiment, we will ferment kombucha, a simple mini-ecosystem that
converts sugared tea into a tart, fruity, effervescent beverage. The origin of kom-
bucha, like many other fermented foods and beverages, is largely unknown; ferments
in their various forms have been exploited for their preservative, flavorant and bio-
chemical effects across the world since time immemorial.
Kombucha is created by brewing tea, typically black tea (a preparation of the
shub Camellia sinensis), adding sugar and inoculating the sugared tea medium with
a SCOBY/mother/mushroom/fungus. This many-named starter is a community of
both yeast and bacteria that live together in a cellulosic mat called a pellicle. Some
brewers are particular about saying the “SCOBY” refers to the liquid rather than the
pellicle itself. However, we will use SCOBY and pellicle interchangeably here. Over
the course of 1-3 weeks, the community grows additional structured pellicle as well
as a planktonic suspension in the liquid medium. Community metabolism is aerobic
and the culture is not sealed. Generally, metabolism involves conversion of sugars
into alcohol (predominantly by yeast) and conversion of sugar/alcohol into acids by
bacteria, giving the beverage its tart, fruity taste. Carbon dioxide is released during
the process, lightly carbonating the beverage. Final alcohol concentrations rarely ex-
ceed 0.5%; below this threshold, a beverage is considered non-alcoholic by the Food
and Drug Administration.
So, in brewing kombucha, we observe the consequences of Baas Becking’s no-
tion of “Everything is everywhere, but the environment selects”. Fermentation in
the medium produces a specialized microenvironment that selects for growth of the
kombucha community, while excluding all other environmental comers.
In the spirit of exploration in biology, we will be trying something new this year
with kombucha brewing. While the traditional drink is made by fermenting tea, a
2Figure 1: A culture of kombucha, the floating disk (pellicle) is made of cellulose
and harbors the microbes responsible for fermentation. Taken from here.
pellicle might be able to live and ferment in other liquids. This has not been exten-
sively studied in the literature. During our first class (on 3/31/21), we will discuss
the various liquids you, the Bi 1x students, are interested in using for the fermenta-
tion. Some first cut ideas the TAs have considered are lemonade, orange juice, maple
syrup, coffee, or caffeinated canned drinks. If you are choosing an experimental liq-
uid, email Liana (lmerk at caltech dot edu) before brewing for us to figure
out a solution for viscosity/sugar issues. As with our class 2 years ago, we will have
a couple of students ferment in sugar water as a comparison to tea fermentation.
Figure 2: A 400x microscope image of the pellicle. Taken by Benjamin Wolfe,
link here.
32.4 16S sequencing and analysis of microbial populations
One of the main ways microbial ecologists identify microbes in complex commu-
nities is with a rRNA sequencing method called 16S sequencing. All archaea and
bacteria have the approximately 1500 bp 16s rRNA gene in their genome. Within the
gene, there are nine variable regions interspersed throughout the highly conserved
sequence, and these variable regions are the target of sequencing, as they can indi-
cate divergence between species. We will be sequencing the V4 and V5 regions using
the 515f and 926r primers described here.
The SCOBY you received in your kits were cut from the same mother SCOBY.
DNA from this mother SCOBY and mother kombucha will be extracted by the TAs,
and it will be the “time 0” of our experiment. Each of you will then ferment your
kombucha at home and send back a bit of your SCOBY in mid-April (about 10 days
of fermentation). By having a beginning and ending time point, we will be able to see
how these different environments change the microbes in the living SCOBY.
3 Protocols
3.1 Creating tea or liquid medium (Wed 3/31/21, week 1)
We will construct different media for fermentation, with a majority of the class
fermenting in the tea they have at home, and others trying the experimental liquid.
The “time 0” pellicle was fermented in caffeinated green tea for two passages (about
one month). It was then split into 30, and placed into a 1/2 previous batch and 1/2
new batch of caffeinated green tea. Note that storing in plastic is not ideal for the
pellicle long term, which is why we are transfering to glass on the first day of class.
In class, we will share a google sheet to list the type of tea or liquid you have chosen for
fermenting in. You may follow along in class on Wednesday or watch the recording.
Follow the protocol below to create your kombucha medium.
1. Wash your mason jar with soap, ensuring that ALL soap has been removed.
Soap will kill your microbes.
2. Begin heating about 700mL water using your method of choice. You may use
the mason jar to measure, it has mL markings on one side.
3. Measure 1/2 cup of sucrose (white table sugar) into your jar. The mason jar
contains cup markings on one side. If you do not have measuring cups, mea-
sure 1/2 way to the 1 cup marking.
4. Unless you are running the “sugarwater” control, add 1 teabag of the appro-
priate type to your jar.
5. With caution to avoid being burned, measure 500mL of hot water from the
water heater and add it to your jar. Stir the water gently until sugar is dissolved.
46. Steep your tea for 15 minutes, stirring gently every 5 minutes.
7. At the end of 15 minutes, remove your tea bag and let the solution cool for
at least 40 minutes. You should be able to hold the mason jar to your wrist
without burning yourself (be careful when testing!!).
8. When the tea has cooled, carefully pour your entire starter tupperware (about
4 oz) into the tea. If you have a paper towel or a (clean!) coffee filter, place
one over the lid and use the rubber band around the lid to secure in place.
Otherwise, place the lid on the jar, but DO NOT SEAL. Keep the mason jar
in a cool place away from direct sunlight as it brews.
Your brew will ferment over 10 days!
IMPORTANT: This is the first (and hopefully last) time we run Bi 1x experi-
ments remotely, and we have never sent kombucha in plastic containers by mail. For
those who locally picked up, your kombucha did not sit in a shipping truck and is safe
to consume. For those we shipped packages to AND to those who are trying experi-
mental brews in non-tea, I urge caution before consuming your brew. Either check in
with Liana by email or Zoom to show me your pellicle. The main risk is mold on the
pellicle, which can be seen in photos here: moldy and non-moldy. I may not be able
to diagnose these problems remotely, so for those interested in experimental liquids,
either cut a piece of your healthy pellicle before subjecting it to new liquids, or do
not sign up for experimental liquids. In the future, continue propagating if you wish,
using the above protocol. Feel free to contact Liana (lmerk at caltech dot
edu) if you have questions on your future brews!
3.2 Sampling the liquid medium (Weekend of April 10, 2021)
We need to introduce as little bias/contamination as possible. Wash your
hands well or wear gloves during this process.
Using a FRESH pipet, transfer 1 mL of the liquid medium approximately in the
middle of the kombucha jar (we want to avoid taking any sedimented cells that might
be dead) into the Eppendorf tube. For those near Caltech, we will arrange a drop off
time on campus. For those who wish to ship back, secure the lid of the tube with tape
and place in a ziplock bag. Place the ziplock in the small shipping envelope. Then,
place that shipping envelope in the large shipping envelope. We will provide you an
address to ship this to.
When the samples arrive to campus, the following protocol will be completed by
TAs. There is a possibility this will be done on Zoom for those interested in watching
the process.
1. Centrifuge at 13,000×g for 1 minute to generate a pellet.
52. Very carefully aspirate and discard the supernatant next to the pellet. Remove
as much medium as possible.
3. Resuspend the microbial pellet in 450uL of buffer MBL from the Powerfood
Microbial DNA kit.
4. To each resuspended pellet, add lysozyme to a concentration of 50ug/mL. In-
cubate 1 hour at 37C.
5. To the lysozyme digested samples, add proteinase K to a concentration of
250ug/mL. Incubate 1 hour at 55C.
6. Set up a negative control with 100 µL sterile PBS (to account for any contam-
ination during extraction).
7. These samples, including the negative control, will now be processed with the
Qiagen Powerfood Microbial DNA kit.
3.2.1 DNA extraction using the Qiagen Powerfood Microbial DNA kit
1. The following protocol, starting at step 4, will be used to extract DNA: man-
ufacturer’s protocol (also at the end of this document).
2. After elution, aliquot 20 µL to a new labeled Eppendorf tube.
3.2.2 Sequencing
The extracted DNA will be sent to Prof. Victoria Orphan’s lab, experts in 16S se-
quencing. They will perform the two PCR preps and prepare the sequencing library.
The sample will then be sent to Laragen for sequencing.
4 Assignment
The assignment for this module is much more open-ended. We are looking at
microbial communities that are largely unstudied. Instead of doing a set of problems,
your Jupyter notebook will be a write-up containing the sections below. Please submit
this notebook both in ipynb format and also as html for ease of reading and grading.
Introduction. Write a one paragraph to one page introduction about the motiva-
tion of the study and what you are looking for with respect to microbial communities
in different kombucha brewing conditions.
Results. Provide results of your analysis about the populations of various species
for the various treatments (type of tea, controls). This can include principle coor-
dinate analysis, relative abundances, etc. This is intentionally open ended, and we
encourage you to explore your data and report your findings. Be sure to comment on
the significance of the results, e.g., What species might be eating caffeine? Are there
any annotations for microbes in the experimental liquids?
6This section is where most of the code cells in your notebook will be. Do not
just show the code cells; you must have ample text in the markdown cells explaining
what you are doing.
Discussion. Here you can expand on the results with any speculations you may
have on why you saw the results you did. Choose at least one microbe annotated
at the species level, in any experimental condition, and discuss literature about this
microbe. e.g. Is it usually found in specific locations? Is it surprising that it is found
in kombucha? You may also posit other hypotheses to test and propose experiments
to test them.
7You can also read
