Research Paper

Observing Microorganism in Making

Tempeh

Angelica Claudia
Eugenia Carisa
9I
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 Preface
First of all, we are very thankful to our teachers that we are given a
chance to do this making tempeh experiment. Through this experiment, we aim at
focusing mainly on the microorganism in making tempeh by using microscope
that is supplied in school. The purpose of this experiment is to know how tempeh
is made with the help of microorganism. We wrote this research paper so that our
viewers will be able to understand what we were working on.

We also very grateful to our mentor who had helped us. We with our
opened hand, ready to receive your comments and suggestion so that we could
learn it and be better in the next opportunity. At last, we hope that our research
paper may help the readers to increase their knowledge and know the use of this
experiment that we conduct through the information that we gave in this research
paper.

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 Abstract
The topic of this research paper is about observing the microoganism in
making tempeh. This observation has the purpose of knowing the structures of the
fungi in tempeh and knowing how the fungi works in the process of making
tempeh. The problem is about the difference of texture in tempeh by using
different amount of tempeh starter to make tempeh. To solvetheproblem, the
writers conduct an experiment by making two tempeh. The process of making
tempeh is by soaking the soybean first then boiled it and put it into the transparent
plastic bag that will be given some tiny holes. The writers predict that the process
of making tempeh will be done by three days.

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 Table of Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5

Basic Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7

Research Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Table of Figures
Figure 1.1 [Rhizopus Oligosporus] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Figure 1.2 [Soybean]
Figure 1.3 [Measuring soybean]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Figure 1.4 [Soaked soybean]
Figure 1.5
Figure 1.6 [Soybean is soaked]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Figure 1.7 [Tempeh starter given to the soybean]
Figure 1.8
Figure 1.9
Figure 2.0
Figure 2.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Figure 2.2 [Day 1 of making 1st tempeh]
Figure 2.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Figure 2.4
Figure 2.5
Figure 2.6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Figure 2.7
Figure 2.8
Figure 2.9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Figure 3.0
Figure 3.1 [Day 4]
Figure 3.2 [Day 1 of making 2nd tempeh]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Figure 3.3
Figure 3.4
Figure 3.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Figure 3.6
Figure 3.7
Figure 3.8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Figure 3.9

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Figure 4.0
Figure 4.1 [Day 4] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Figure 4.2 [Measuring 1st tempeh]
Figure 4.3 [Measuring 2nd tempeh]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Figure 4.4 [Cutting 1st tempeh]
Figure 4.5 [Cutting 2nd tempeh] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Figure 4.6 [Observed fungi]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24

Table of Diagrams
Diagram 1.1 [Fungi classification]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Diagram 1.2 [1st tempeh observation] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Diagram 1.3 [2nd tempeh observation]. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Diagram 1.4 [Weight comparison between both tempeh]. . . . . . . . . . . . . . . . . . 22

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 Introduction
Tempeh is a famous Indonesian traditional food that is made from
soybean. Tempeh can be made by other ingredients too, such as blackbeans,
black-eyed peas and chickpeas or by using grains such as, brown rice, barley or
millet, but tempeh nowadays is mostly made by soybean.The writers are
interested with this topic because tempeh is one of the food that is produced with
the help of fungi.

Tempeh is the product of the activities of fungi or usually known as

Rhizopus Oligosporus. Making tempeh needs fungi, people usually know this as
tempeh starteras it contains Rhizopus Oligosporus. By this process of changing
the soybean into tempeh, it makes the tempeh healthier and more delicious
because the taste and the aroma of the soybean is changed when it becomes
tempeh.

The purpose of conducting this experiment so that the writers may know
the role of fungi in the process of making tempeh. Through this experiment, the
writers can know the structures of the fungi in tempeh by using microscope.

The statement of problems

1. Do the amount of tempeh starter affect the final result of tempeh?

2. How many days are needed until the tempeh is ready?
3. Do the temperature changes obstruct the growth of tempeh?

For the variables, there are three types of variables such as independent,
dependent and controlled variable. In doing the experiment, the writers will make
two tempeh with different amount of tempeh starter given to the soybean which
means the amount of tempeh starter given will be the independent variables. As
the writers wanted to know what the difference of both tempeh result is, the
writers will observe it by looking at the texture of tempeh and the mass of the
soybean before and after it becomes tempeh (dependent variable). So, the one that
will stay the same is the amount of soybean used to make tempeh, means it is the
controlled variable.

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 The writers expect that 3 days is enough to obtain the final result of the
tempeh. Since the process of soaking the soybean takes 12 hours, because the
writers think that it is enough for the de hull process. Then, using another 24 – 48
hours to keep the tempeh that has been given tempeh starter in room temperature.
Therefore, the writers predict that 3 days is enough for the mycelium to form thick
white layer and to be bounded firmly like a ‘white cake’.

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 Basic Theory
Tempeh is made out of fungi. Fungi is an eukaryotic organism. An
eukaryote is any organism whose cells have a cell nucleus and other organelles
enclosed within membranes. Fungi don’t have any chlorophyll to do
photosynthesis so, fungi absorbs organic substance from its environment. This is
called as heterotrophic. Before absorbing, the enzyme produced by the fungi
makes the organic substance simpler. (Anonymous A).

As heterotrophs, Fungi can be :

1.Parasites, parasites obtain nutrients from its host that causes the host to be
harmed
2.Mutualistic symbols, the fungi also obtains nutrients from its host but it provides
benefits to that host unlike parasites
3. Saprobes, this type of fungi obtains nutrients form nonliving organic material or
the remains by-products of organisms. The mycelium surrounding a dead fly on a
windowpane or the fungi that cause rot in wood are examples of this saprophytic
mode of nutrition. (Anonymous A )

Four main of fungi classification :

Class Structure Example

The mycelia are divided into Produce tempeh (Rhizopus
three parts : the rhizoids on the Oligosporus), black bread
below surface, sporangiophores mold (RhizopusStolonifer)
Zygomycota above the surface and stolon that
connects rhizoids and there is
also sporangiophores around it.
(Sparknotes, 2017)
The mycelia can produce two Morel mushroom
Ascomycota types of reproductive structures. (Study.com)
(Sac Fungi) For asexual reproduction, they
produce structure called

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 conidiophores and for sexual
reproduction, they produce
structure called gametangia. Most
of Ascomycota produce a fruiting
body. (Sparknotes,2017)
The mycelia grows under the Mushroom, Puffballs, and
surface. The plasmogamy join Shelf fungi
Basidiomycota
and produce binucleate dikaryotic
(Club Fungi)
hyphae that form the fruiting
body. (Sparknotes,2017)
Resemble ascomycetes, but their Cheese, sausage
reproductive cycle has never been (PenisilliumChrysogenum).
Deuteromycota
observed due to definite lack of (Enrico,2015)
(Imperfect Fungi)
sexual reproduction, that’s why
they are called imperfect fungi.
(Diagram 1.1)

To describe the structure of fungi, there are Hyphae and Mycelium

(Ardi,2016). Hyphae is thread-like structure of very fine, colorless threads that
makes up the body of a fungus. While Mycelium is a body of fungi that made up
of many hyphae.

The tempeh is made by soaked and cooked soybeans that are inoculated by
mold which hydrolyzed the soybeans constituents and it affects the texture, flavor
and aroma of the product (KAHachmeister1993). It is easy to make tempeh and it
only takes at least 24 hours to grow the Rhizopus Oligosporus onto soybeans, but
to keep it may be 36 to 48 hours (Veganlovlie2016). Usually tempeh is wrapped
with banana leaves but in developed countries they use transparent plastic that is
given some tiny holes to allow air flow since too much air will make the mold
produce a lot of spores (EGulick2014).

Oxygen is needed for the growth of mold, because in general most of

Rhizopus do aerobic respiration which needs oxygen therefore, heat is produced.
Too much oxygen will make more heat produced that will obstruct the growth of

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the fungi. When the heat reached the maximum heat, the fungi will die.
(Anonymous2014). Tempeh is made by giving the soybeans tempeh starter that
will grows the fungi (Rhizopus Oligosporus). Usually the amount of tempeh
starter for a 1 kg of soybeans are 2 grams of tempeh starter.
(OrganicCultures2010)

Indicators that tempeh is ready :

1. The soybean is already inoculated with the white molds.

2. The color looks like a boiled soybean (not too bright and not too
dark).
3. The soybean will not be ruined when it is sliced.
4. When we touch the tempeh, it is not too hot, because if it is too hot
the tempeh will be spoiled faster.

Indicators that tempeh is not good :

1. Fail tempeh can be known by its aroma and texture. If the aroma is
sour, it means the tempeh is already old
2. The color of the tempeh is not bright and fresh, the color must be
brownish or greyish
3. Tempeh that is wet and slippery means that it cannot be consumed
anymore
4. If the tempeh is hot and the packing is wet like it is sweating, it
means that the fungi is still working as it will be smelly because
the fungi works too long (Figure 1.1)

Since mold produce spores, the spores will start

to grow and the mold will cover the soybeans. The type
of microorganism that involved in the production of
tempeh is called Rhizopus Oligosporus. Rhizopus
Oligosporus is the main fungi in the process of making
tempeh that is classified as Zygomycota which is a small
group in the fungi kingdom and it can reproduce asexually

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or sexually, in a process called conjugation. Conjugation is the temporary fusion
of organism. (Boundlesstextbook)

Every fungi in the process of making tempeh will produce enzyme

(Ika,2016). Rhizopus Oligosporus can produce protease and Lipase enzymes that
makes tempeh has the unique flavor and aroma. Protease breaks down protein into
amino acid while lipase breaks down fats into fatty acids and glycerol. The good
temperature for the mold to grow is around 25-27oC. (SirOssiris2011)

Since the 12th to 13th century, the soybean has been recognized in
Indonesian island of Java. In the 17th century, the Chinese introduced the tofu-
making industry in Java. The legend says that tempeh was discovered by accident
when discarded the soybean residue caught the spores and grew a certain whitish
fungi that was to be edible

Tempeh is becoming popular, and today more and more grocery stores
hold tempeh products. This is because tempeh is known to reduce cholesterol,
increase bone density, reduce menopausal symptoms and promote muscle
recovery. In addition to these amazing benefits, tempeh has the same protein
quality as meat and contains high levels of vitamins B5, B6, B3 and B2 (Dr. Axe,
2017). Minerals and another uncommon contents are also contained in tempeh.
This is why tempeh is called as one of the healthiest foods in the world

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 Research Method
Materials :

1. Soybean 300Grams
2. Tempeh starter (1/2 teaspoon and 1 teaspoon)
Based on own measurement : 1 teaspoon equals to 2 grams

Apparatus :

1. Pan
2. Transparent plastic bag
3. Banana leaf
4. Stapler
5. Process :
1. First, take the soybean and pick the good soybean then wash it until its
clean.

(Figure 1.2) Washed

soybean

2. Second, measure your desired amount of soybean

(Figure 1.3) measuring the

soybean
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 3. Next, soak the soybean for around 12-24 hours until we can get rid of the
skin.

(Figure 1.4) Soaked

soybean

4. Fourth, after soaking the soybean, we rid off the skin.

(Figure 1.5)

5. Fifth, boil the soybean using pan until the soybean is tender. After that, dry
it.

(Figure 1.6) Soybean is

boiled

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 6. Sixth, add the tempeh starter to the soybean sufficiently and mix it. Then,
put all of the soybean into the plastic bag.
(Figure 1.7) (Figure 1.8)

7. Then, close the plastic bag firmly using stapler and make some tiny holes
approximately 1 inch interval. (Figure 2.0)

(Figure 1.9)

½ teaspoon of tempeh starter 1 full teaspoon of tempeh starter

(1 gram) (2 grams)
8. Finally, put the soybean in room temperature.

(Figure 2.1)

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We will make 2 tempeh by using different amount of tempeh starter. After adding
the tempeh starter to the soybean, the process of fungi growth will start. Then
after it is done, we will compare the texture of both tempeh and their mass before
and after. The amount of tempeh starter used is increased to make it easy to be
differentiate, based on basic theory the standard one is 2 grams of tempeh starter
for 1 kg of soybeans, but here we are going to use 2 grams of tempeh starter for
300 grams of soybeans and 1 grams of tempeh starter for other 300 grams of
soybean to make it easy to be compared since what we are going to observe is do
the amount of tempeh starter affect the final result of making tempeh.

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 Results and Analysis
The result from our experiment proved that the amount of tempeh starter affects
the result of the tempeh. As we have stated before, our main focus is to know do
the amount of tempeh starter affect the result of the tempeh. Therefore, we made
two tempeh with different amount of tempeh starter. We observe the tempeh until
it is spoiled while the tempeh with 1/2 teaspoon of tempeh starter should be ready
at day 3 , 17:00. This is the result that we conduct :
½ teaspoon of tempeh starter, we started making the tempeh at 14:00 on
September 7th
(Diagram 1.2)

Time Figure Description

There were no any

Day 1
changes occurred to
14:00
the soybean.

(Figure 2.2)

The water vapor

Day 1 came from the
17:00 soybean started to
show up.

(Figure 2.3)

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 The water vapor
Day 1 increased and the
21:00 soybean started to be
warm.

(Figure 2.4)

The water vapor

Day 2 increased and the
06:00 soybean became
shaped and chill.

(Figure 2.5)

The water vapor

decreased and the
Day 2
soybean became
17:00
warm and even more
shaped.

(Figure 2.6)

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 The water vapor
decreased and the
Day 2 soybean became
21:00 chill again and some
mold began to show
up

(Figure 2.7)

The soybean was

Day 3 evenly inoculated by
06:00 the mold and
became warm

(Figure 2.8)

This was the other

side of the tempeh
and it was also
Day 3
inoculated by the
17:00
mold but not as even
as the other side of
the tempeh

(Figure 2.9)

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 There were no changes
Day 3
occurred to the
21:00 soybean.

(Figure 3.0)

The tempeh became

Day 4
spoilt because it
09:00 produced smelly scent

(Figure 3.1)

We observed the tempeh until it is spoiled while the tempeh with 1 teaspoon of tempeh
starter should be finished on day 2, 21:00. This is the result that we conduct :

1 teaspoon of tempeh starter (Diagram 1.3)

Time Figure Description

(Figure 3.2)

There were no any

Day 1
changes occurred
14:00
to the soybean.

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 The water vapor
Day 1
came from the
17:00
soybean started to
show up.

(Figure 3.3)

Day 1 The water vapor

21:00 increased

(Figure 3.4)

The water vapor

Day 2 increased and the
06:00 soybean became
shaped

(Figure 3.5)

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 The soybean were
Day 2
evenly inoculated
17:00
by the mold

(Figure 3.6)

The soybean were

Day 2 more evenly
21:00 inoculated by the
mold.

(Figure 3.7)

The tempeh
Day 3
produced awful
06:00
smell.

(Figure 3.8)

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 Unwanted bacteria
began to obstruct
Day 3
the growth of
17:00
Rhizopus
Oligosporus

(Figure 3.9)

The tempeh was

Day 3
the same as the
21:00
previous one.

(Figure 4.0)

The tempeh
Day 4 became more
09:00 smelly and became
spoiled.

(Figure 4.1)

After conducting the result from this experiment, we conclude that more tempeh
starter makes the growth of the fungi become faster because the tempeh starter
were spread evenly and it will produce a huge amount of spores that will make the
soybeans to be inoculated faster. The result showed that the one with more tempeh

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starter produced the smelly scent faster than the one with little tempeh starter,
since we kept the tempeh for 4 days, which is too long, the fungi keeps respirate
and produce more heat which obstructed the growth of Rhizopus Oligosporus and
made the tempeh became spoilt.
Another result that we found show that both of the tempeh have the different
mass. (Diagram 1.4 shown below)

Before After

½ teaspoon of tempeh starter (Figure 4.2 shown below)

(300 gram) (300 gram)

1 full teaspoon of tempeh starter (Figure 4.3 shown below)

(300 gram) (310 gram)

Based on the diagram above, both of the initial mass were 300 gram, there are
mass difference for the second tempeh, as the amount of tempeh starter used were
more than the first one. The second tempeh is heavier than the first tempeh, more
tempeh starter are given, the more the mold grow and spread widely inoculating
the whole soybean evenly which making it heavier. While the first tempeh
actually have to be in the different mass because the growing of the mold should
have increased the mass of the object but unfortunately the scale didn’t show any
difference.
There might be a probability that support the statement saying that before and
after have the same mass :
1. First, the scale that was used was not that accurate since it is a normal
scale that doesn’t measure very accurately.

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 2. Second, there might be an error in seeing the measurement as not all of
numbers are in the scale, only certain numbers in sequence.
Anomalies were found during this experiment. First, the tempeh with 1 teaspoon
of tempeh starter looked great outside but inside, the mold didn’t cover the gap
between the soybeans .While the tempeh with ½ teaspoon of tempeh starter
looked awful outside because there was a part on the tempeh which was not
covered by the mold at all, it can be caused by some factors. To make it clearer,
here are some pictures of our tempeh we made ourselves.
This is the result of tempeh with ½ teaspoon of tempeh starter. (Shown in the
figures below)
(Figure 4.4)

While the tempeh with 1 teaspoon looked great outside, but inside the mold didn’t
cover the space between the soybeans well.
This is the result of tempeh with 1 teaspoon of tempeh starter. (Shown in the
figures below)
(Figure 4.5)

Another anomaly that we found was that our tempeh was too wet, proved by the
water vapor shown in the pictures because the temperature and the location where
we placed the soybean didn’t support this process.

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We can say that the tempeh experiment was spoilt and was not evenly inoculated
by the mold due to some factors that didn’t support our experiment :
1. The tempeh was too wet and the growth of the mold was not even.
This was caused by the temperature changes. It was too wet because the
temperature was too high. The high temperature will obstruct the growth of
the mold, it will caused some water vapor to appear making the tempeh wet so
the mold couldn’t really evenly grow due to humidity which there will be less
air that travel making the spores not evenly spread so there will be some parts
that don’t get inoculated by the mold. Tempeh mold don’t grow in low
temperature because it needs humidity too, but too much humidity can
obstruct the mold growth too.
2. The holes were too small and little
The holes were too small so it affected the exchange of oxygen. Some holes
should be made by using nails to make the holes bigger instead of toothpick,
but not too many holes because too much oxygen can make the tempeh
overheated.
The main topic of our experiment is to observe Rhizopus Oligosporus and its
structures. This is the Rhizopus Oligosporus we captured.

(Figure 4.6 shown below)

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 Conclusion
The investigation was about observing the fungi found in the process of making
tempeh, Rhizopus Oligosporus but of course we got to make the tempeh before we
can observe the fungi through microscope. Based on our statement of problems,
which was the amount of tempeh affected the final result, the answer is yes. The
tempeh with 1 teaspoon (2 grams) of tempeh starter grew its mold faster than the
tempeh with ½ teaspoon (1 grams) of tempeh starter because more tempeh starter,
the fungi starts to grow and produce more spores making the mold to grow faster.
Our expectation stated that we were taking 3 days to obtain the final result of the
tempeh but we observed it until day 4 until they were spoilt to know more what
might be the factor that caused the tempeh to become spoilt. Tempeh need to be in
a certain temperature so that the molds can grow evenly and become a good
tempeh. The answer for the factor that caused tempeh to become spoilt because
there are temperature changes that somehow can make the fungi die too producing
an awful smell.
The importance of this experiment which is observing the fungi – Rhizopus
Oligosporus is to know the structures of the fungi not only by looking for the
pictures through internet but directly observe it by microscope and know its
structures that is proven by our drawing (Shown in figure 4.6).

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 References
http://cara-membuat-tempe.blogspot.co.id/2012/10/mengenal-jamur-tempe.html
http://www.sridianti.com/perbedaan-antara-hifa-danmiselium.html
https://www.ncbi.nlm.nih.gov/pubmed/8267862
http://blogs.evergreen.edu/fungalkingdom/rhizopus-oligosporus-the-fungus-that-
enhances-plant-foods-into-tempeh/
https://biologimapiha.blogspot.co.id/2016/07/proses-pembuatan-tempe.html
https://lordbroken.wordpress.com/2011/07/09/fermentasi-tempe/

http://arti-definisi-pengertian.info/pengaruh-faktor-lingkungan-terhadap-jamur-r-
oligosporus/

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