Using Fermentation to Observe Ecological Succession

Francisco Diaz
Assisted by Catrina Shurtleff
12pm-2pm Tuesday, Thursday
November 18, 2016

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Abstract:
Ecological succession is the change in species found in an ecosystem over time due to
changes in the environment (Gibbons, 2015). To understand how ecological succession takes
place; sauerkraut was made using a homemade recipe which will be the ecosystem for the
bacterium. Ecological succession will be observed when fermenting cabbage and cucumbers. To
observe ecological succession take place, sauerkraut was made in a mason jar using a recipe
from Kitchn: How to Make Homemade Sauerkraut in a Mason Jar. A sample of 3 mL was used
to creat a serial dilution was made according to the colony formation found on the plate. From
the results, it is seen how there was not much growth in anaerobic or microaerophilic bacterium
from day 0, this was observed in both ecosystems. It was also hypothesized that the pH
concentration will drop over time and the most abundant organisms that will be found in the
ecosystem will be Lactobacillu, since it has a higher acidic tolerance (McDonald, 1990) and
Coliform bacteria. The prediction of the Pseudomonads organism being scarce was correct for
the pickled recipe, but was not consistent win the sauerkraut recipe.
Introduction:
Fermentation has been documented to be used throughout history to preserve food as well
as the taste. Fermentation in food processing involves the process of converting carbohydrates to
alcohol or organic acids using microorganisms. This process requires the chemical breakdown of
a substance by bacteria, yeasts, or other microorganisms within anaerobic conditions. Thanks to
fermentation there are products such as beer, wine cheese, pepperoni, soy sauce, yogurt, and
other pickled products such as sauerkraut. With the help of zymology (The study of biochemical
process in fermentation), it is understood that bacteria biochemical pathways to convert glucose
into alcohol and acids in the absence of air.

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Ecological succession is the change in species found in an ecosystem over time due to
changes in the environment (Gibbons, 2015). To understand how ecological succession takes
place; sauerkraut was made using a homemade recipe which will be the ecosystem for the
bacterium. Ecological succession will be observed when fermenting cabbage, during
fermentation the environment will become more acidic. According to Ecology of Fermented
Foods the organisms that are most common for fermenting cabbage are Coliform bacteria,
Leuconostoc, and Lactobacillus (Scott & Sullivan, 2008). It is hypothesized that there will be a
scarce amount Pseudomonads and other aerobic organisms on the last day, compared to the
beginning of the fermentation process since it is very common for Pseudomonads to be obligated
aerobes. This observation was made using Bergies Manual. As the environment becomes more
acidic, it was also hypothesized that the pH concentration will drop over time and the most
abundant organisms that will be found in the ecosystem will be Lactobacillu, since it has a higher
acidic tolerance (McDonald, 1990) and Coliform bacteria. Not only it is assumed that
Lactobacillus be most abundant it will also grow at a slow rate as the environment becomes more
acidic its growth will become more favorable, both in the ferment sauerkraut, and cucumber
ecosystem.
Materials and Methods:
To observe ecological succession take place, sauerkraut was made in a mason jar using a
recipe from Kitchn: How to Make Homemade Sauerkraut in a Mason Jar. This recipe requires 3
pounds of cabbage, 5.5 Tablespoons of kosher salt, and it was a dry recipe. A few modifications
were made to the recipe since 3 pound was a large amount to work with. The first step was to
figure out how much cabbage will be used; the empty mason jars weight was recorded and then
it was stuffed with cabbage and weighed once again to identify the difference in weight and

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determine the amount of cabbage necessary. Using the weight information of the cabbage you
can create proportion that will satisfy the salt requirements. Once the amount of salt was
determined it was placed in the jar with the cabbage; since this was a dry recipe distilled water
was added to the mason jar until it covered the cabbage completely. 73g of cabbage, 5.104g of
salt, and 322 mL of distilled water were used for the fermentation of sauerkraut. For future
purposes the proportion for turning grams of cabbage to lbs.
1lb cabbage
xlbcabbage
=
=.16 lbcabbage
453.59 g cabbage 73 g cabbage

table spoons

grams

proportion for determining the number of

3 lb cabbage .16 lb cabbage

=
=.293 tbs . proportion made to convert tbs into
5.5 tbs salt
x tbs salt

1 tbs salt
.293tbs salt
=
=5.104 g salt .
17.06 gsalt
xg salt

The pickled recipe was made by Shannon Stonger, 2016. It consisted of boiling water for a
minute to kill any organism that were found in the water. The cucumber recipe required 21 grams
of salt, 2 dill leaves, 2 bay leaves, 2 garlic gloves, and 400 mL of boiled water. All the ingredient
were placed in the mason jar and both of the fermented items were fermented for the same
number of time.
Samples from the fermented cabbage and cucumber were taken on days: 0, 5, 7, 12, and
14. The samples were taken using a sterile syringe and needle. On each of those days the rubber
septum of the lid will be penetrated and three mL of the solution was taken from the jar. Every
time a sample was taken from the jar, it was replaced with a brine solution. From the sample, a
serial dilution was made according to the colony formation found on the plate. What was left

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from the original 3 mL was used to measure the pH of the fermented product using pH strips for
each day that the sample was taken from the jar.
The plates that were used to identify genera of the organisms found in the ecosystem are
as followed: WN5 which is a differential medium containing 5% Na Cl, this plate selects for
Pediococcus, Pseudomonads agar (PS) which selects for Pseudomonads, Lactobacillus
-Streptoccoccus Differential agar (LSD) which selects for both Lactobacillus and Streptoccoccu,
a plat that would Select for entrics (EC), Trypicase Soy Agar (TAS) non-selective medium that
will have an array of organisms growing in this medium. The list for the type of plate and
condition is: PS aerobic, LSD aerobic, TSA aerobic, EC aerobic, WN5 placed in microaerophilic,
WN5 anaerobic, and a TSA in anaerobic conditions.
The dilution scheme and colonies count can be seen in the result section below. The test
protocols for the biochemical test were obtained from Microbiology Laboratory Theory
& and The Ecological Succession during Food fermentation lab manual provided by
Dr. Bean 2016, was consulted.
Results:
The morphology of the bacterium found growth in each of the plates for the cabbage
fermentation is described in table 1. Table 1 also indicates how there were different
morphologies on day 0 for each plate, but as time progressed some of the morphologies were
lost. The dilution scheme that was made was based on the growth that was observed on each of
the plates. Table 2. Contains the number of colonies that grew on each of the plates and the
dilutions scheme to obtain that colony count for the cabbage. The pH over time is shown on table
3 for the cabbage. On day 0 there was not much growth; the aerobic plate contained the most
growth. The pH went from almost neutral to acidic over the 15-day period in which data was

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recoded. The most dramatic pH change in the cabbage fermentation was noticed on day 5; the pH
dropped from 6.8 to 6.5. Figure 2 illustrates how all the bacterium that were grown on the pates
had growth, apart from the anaerobic WN5 plate did not show any growth over the 15 days it
took the sauerkraut to ferment. Figure 2 also illustrates how Pseudomonads concentration
dropped on day 7 and quickly tripled in concentration found in the sauerkraut ecosystem on day
12. The concentration shown in figure 2 for all the organism is constantly increasing except for
the Pediococci that was anaerobic, and Pseudomonads.
The cucumber data indicated that the Pseudomonads concentration declined after day 5,
along with the aerobic organisms this is shown in figure 3. The pH for this ecosystem drastically
changed after day 0. As seen on table 4 the pH was 6.0 and it dropped to 4.5, this pH remained
Type of Plate
Pediococci
(microaerophil
ic)

Pediococci
(anaerobic)

Pseudomonads
(aerobic)

Lactobacilli
(aerobic)

All (aerobic)

All (anaerobic)

Enteric (aerobic)

Day 0
Day 5
0
22
CFU/m
(101)
L
1
(10 )
0
CFU/m
L
(101)
1
1
(10 )
42
CFU/m
L
(101)
334
CFU/m
L
(101)
0
CFU/m
L
(102)
55
CFU/m
L
1
(10 )

0
CFU/m
L
(101)
3206
CFU/m
L
(101)
1679
CFU/m
L
(102)
5678
CFU/m
L
(102)
2578
CFU/m
L
(101)
3836
CFU/m
L
1
(10 )

Day 7
100
CFU/m
L
(
101
0
CFU/m
L
(101)
23
CFU/m
L
(102)
792
CFU/m
L
(103 )
3836
CFU/m
L
(103 )
6400
CFU/m
L
(103 )
1200
CFU/m
L
3
(10 )

Day 12
350
CFU/mL
(101)

Day 14
3439
CFU/m
L
(102)

0 CFU/mL
(101)
53
CFU/mL
4
(10 )
216
CFU/mL
(104 )
3528
CFU/mL
(104 )
1640
CFU/mL
(104 )
2788
CFU/mL
4
(10 )

0
CFU/m
L
(101)
119
CFU/m
L
(106 )
66
CFU/m
L
(106 )
61
CFU/m
L
(106 )
77
CFU/m
L
(106 )
32
CFU/m
L
6
(10 )

relatively constant from day

5 to day 14, the pH never
dropped below 4.3 refer to
figure 4.

Table 1. This is table

indicate the dilution that
was used to plate and the
number of colonies found
on each of the selective
plate based on their genera,
since it was not possible to
count colonies over 1000
the plate was divide into 4
sections and only once of
those sections was counted
and multiplied by four.
To determine the
CFU/mL calculation 1
was used.

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Day in which
the pH was
read
0
5
7
12
14

pH
Concentration
6.8
6.5
6.3
6.1
6

Calculation 1:
Number of colonies per mL plated
=CFU /mL
Total dilusion facter
66 colonies
7
=66 x 10 CFU /mL
6
10

example

Table 2. Recorded pH on each day of observation of cabbage,

recorded with pH strips.

Changes in the Acidity of the Ecosystem due to Fermentation

Figure 1: Indicates the change in pH over time for the fermentation of cabbage.

Change in pH Throughout the Fermentation Process in C. sativus

Figure 2. Graph of the change

in pH over time throughout the course of cucumber fermentation.

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Day

pH

6.0

4.5

4.4

12

4.3

14

4.3

Table 3. Recorded pH on each day of observation, as recorded with pH strips of pickles.

Ecological Sussion In Sauerkraut Fermentation

Pediococci Microphilic

Pediococci Anaerobic

Pseudomonads

Lactobacillus and Streptococcus

All Aerobes

All Anaerobes

Entrics

Figure 3. Graph of the change in the concentration of microorganism over time throughout the
fermentation process of cabbage, in CFU/mL. The y-axis of the figure is adjusted on a base-10
logarithmic scale.

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Growth By Ecological Succession in C. sativus Fermentation

Pediococci (microaerophilic)

Pediococci (anaerobic)

Pseudomonas

Lactobacilli

Enterics

All aerobes

All anaerobes

Figure 4. Graph of the change in microflora growth over time throughout the process of
cucumber fermentation, in CFU/mL. The y-axis of the figure is adjusted on a base-10
logarithmic scale.
Day
Organism

12

14

Pediococci (Microaerophilic)

5.00E+01

1.75E+06

3.50E+06

1.58E+07

2.80E+07

Pediococci (Anaerobic)

1.00E+00

1.0E+05

2.00E+05

1.00E+00

2.50E+04

Pseudomonas

9.90E+02

4.95E+02

1.00E+00

1.00E+00

1.00E+00

Lactobacilli

2.26E+03

2.15E+07

4.30E+07

7.60E+06

8.90E+06

Enterics

4.20E+03

2.1E+03

1.00E+00

1.00E+00

1.00E+00

All aerobes

5.00E+04

3.45E+07

6.90E+07

1.28E+07

1.32E+07

All anaerobes

3.45E+03

2.55E+07

5.10E+07

1.43E+07

1.50E+07

Table 4. Data from daily observed counts converted into original cell density (OCD) in
CFU/mL. OCD for day 5 are estimated, since there was no quantifiable raw data for that day.
Organism genera are named based on what each media selects for, and therefore which
organisms are present on the various plates.

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Discussion:
From the production of sauerkraut ecological succession was observed. The expectations
for the ecologic successions were that the Pseudomonads population should have declined over
time, along with any aerobic bacterium as the ecosystems oxygen is consumed. The hypothesis
that was formed was that the pH concentration will drop over time and the most abundant
organisms that will be found in the ecosystem will be Lactobacillus, and Coliform bacteria. The
pH was also expected to drop. This was all based on the assumption that the aerobic bacterium,
that rely on oxygen to be able to ferment, would no longer be able to do so as the oxygen levels
drop. As the fermentation process occurs the environment would become more acidic from the
acid by-product that is produced during the bacteriums formation. This would create a favorable
environment for Lactobacillus.
From the results, it is seen how there was not much growth in anaerobic or
microaerophilic bacterium from day 0, this was observed in both ecosystems. As time progressed
both ecosystems began to populate the environment, this is indicated in table 1 and 4. There are
differences such as that the concentration of Pediococci anaerobic, and microaerophilic found in
the environment was much greater in the pickled ecosystem and the organism Pediococci was
most abundant in the ecosystem, this is illustrated in Figures 4. In comparison to the sauerkraut
ecosystem, although it had growth of the Pediococci organism, it was not the most abundant
organism found in the environment.; Figure 3, illustrates that the most abundant orgasm was
Lactobacillus. This is similar to the result that were provided by Scott, and Sullivan in the
Ecology of Fermented Foods, they stated that the Coliform bacteria would create the
environment for the Lactobacillus, and most other organisms would not be able to tolerate. There

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were also many anaerobic organisms in the sauerkraut ecosystem. Another difference is that
there was a no growth of Pediococci anaerobic organisms in the sauerkraut environment.
The hypothesis was that the pH concentration will drop over time and the most abundant
organisms that will be found in the ecosystem will be Lactobacillus, since it has a higher acidic
tolerance (McDonald, 1990). There should also be a large population of Coliform bacteria, both
in the ferment sauerkraut and pickled ecosystem, was semi correct. Although Lactobacillus was
the most abundant in the sauerkraut, it was not the most abundant in the cucumber ecosystem. It
was also hypothesized that the environment would become acidic. Both ecosystems did indeed
become acidic, but the pickled environment showed the most dramatic pH change as shown in
Figure 2. Figure 1 illustrates the change in pH for sauerkraut, even though it did become a
slightly more acidic environment the only dramatic change that occurred when the pH changed
from 6.8 to 6.5. There were a few times when pH was hard to read since pH paper was not very
accurate, it could have been giving false readings of the pH in both environments.
The prediction of the Pseudomonads organism being scarce was correct for the pickled
recipe. For the sauerkraut recipe, the organism concentration did decrease, on day 7, when it was
plated on day 12 as illustrated in figure 3 the concentration increased, it also increased on day 14.
This may have been due to the rubber cork being loosened without being noticed until day 12.
This would provide the aerobic Pseudomonads with a favorable aerobic environment allowing it
to reproduce. It was not possible to identify if the fermentation worked since the products were
not allowed to be eaten, but based on the acidity it can be concluded that it worked.

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References:
Gibbons, SM, et al. "Ecological Succession and Viability of Human-Associated Microbiota on
Restroom Surfaces." APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 81, no. 2,
2015., pp. 765-773doi:10.1128/AEM.03117-14.

Christene, Emma. How To Make Homemade Sauerkraut in a Mason Jar Kitchn.

http://www.thekitchn.com/how-to-make-homemade-sauerkraut-in-a-mason-jar-193124
August 16,2016.
McDonald, L. C., H. P. Fleming, and H. M. Hassan. "Acid Tolerance of Leuconostoc
Mesenteroides and Lactobacillus Plantarum." Applied and Environmental Microbiology, vol. 56,
no. 7, 1990., pp. 2120-2124.

Scott, Robert, and Sullivan, C. William. Ecology of Fermented Foods. Rep. Department of
Natural Resources and Environmental Sciences, University of Illinois. Urbana: Human Ecology
Review, 2008. Print.

Bean, Heather Bean. The Ecological Succession during Food Fermentation Lab Manual.
School of Life Sciences Arizona State University. 20016.

Bergamaschi, Julia, Microbiology Lab 302, 2016

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Stonger, Shannon. How to Make Homemade Lacto-Fermented Crunchy Dill Pickles. Health
Impact News. https://healthimpactnews.com/2015/how-to-make-homemade-lacto-fermentedcrunchy-dill-pickles/ July 2015.