FERMANTATION

PRACTICAL REPORT
Project 1: Determination of antibiotic production from actinomycetes isolated
from soil and plant materials

INITIALS AND SURNAME STUDENT NUMBER

J MHLONGO 220005923

M LEMEKE 219173044

BT MANALENG 219165114

OL KAREN 219133980

JUNE 29, 2021

VAAL UNIVERSITY OF TECHNOLOGY
Title

Determination of antibiotic production from actinomycetes isolated from soil and plant
material

Introduction

Soil and plants act as important sources of microorganisms that are of great importance in
science and medicine. Among the microbes isolated from both soil and plants,
Actinomycetes are one of the most important groups that are relevant to scientists and
microbiology laboratories due to their ability to provide many important bioactive substances
that have high commercial value. Actinomycetes are found everywhere in nature and can
also be found in man-made environments, they are found in large numbers in the soil, fresh
waters, lakes, river bottoms, manures and in food products. Actinomycetes are filamentous
gram-positive bacteria, they can be easily differentiated from other bacteria by their
morphology and DNA, which is rich in guanine and cytosine. They are easily identified by
having a high G+C content (>55%) in their DNA (Gonzalez-Franco & Robles-Hernandez,
2009). They are organisms with characteristics common to both bacteria and fungi, yet they
possess distinctive features to distinguish them into a distinctive category (Abbas, 2016).
Actinomycetes have spore forming abilities and form structures of mycelia and ultimately
belong to the order Actinomycetes. They are of extraordinary importance in different areas
such as medicine and pharmaceutical industry, particularly in antibiotic production. They are
the best commonly known source of antibiotics, two groups being Streptomyces and
Micromonospora are two major groups of soil Actinomycetes that are significant sources of
antibiotics. A large number of currently used antibiotics which includes streptomycin,
gentamycin, erythromycin and rifamycin are all products of the soil Actinomycetes isolate
(Jeffrey, 2008).

Isolating or classifying microorganisms according to their characteristics or capabilities

requires correct isolation methods and cultivation. A handful of healthy soil contains
approximately a million different microorganisms or more, therefore serial dilution must be
done to determine how many microorganisms are in each sample. Serial dilution involves
distributing the original sample in equal amounts till it is further diluted, this ensures that the
sample has cells (or colonies) that are easy to work with. Serial dilution is done using dilution
factors that may range from 10-1 to 10-7 depending on how concentrated the original inoculum
is. Dilutions are plated on medium like pour plate and spread plate to further cultivate and
make it possible to study the characteristics, biochemistry and capabilities of the bacteria or
sample in question.
Microorganisms grow inside the tissues of plants, and they survive on minimum resources.
Microbes are found in most of the plants, and they live in a mutualistic relationship.
Actinomycetes are mostly found when one isolates microbes from the plants, they play an
important role in the breaking down of organic matter into nutrients that are easily obtained
and for the production of many secondary metabolites (usually have an important ecological
function like antibiotics and pigments) that are important for pharmaceutical and agricultural
industries.

To be able to isolate microorganisms from plants, the plant sample must be placed on a
specially prepared medium which will allow the growth of the microorganisms. The sample
must be pressed on the agar using a scalpel since the microbes are situated in the leaves.

Gram staining, a differential staining technique is done to differentiate between Gram-

positive and Gram-negative bacteria because of their differences in cell structure. This
technique was developed by Dr Christian Gram in 1884. Gram-positive organisms have thick
peptidoglycan layer meanwhile Gram-negative organisms have a thin peptidoglycan layer.
During a Gram-stain, heat fixation of the slide is done before adding crystal violet for one
minute and gently rinsing the slide with water. Iodine is then added for another minute before
rinsing once again. It acts as a mordant to form a crystal violet-iodine complex. Following
this is decolourization with acetone alcohol for 5 seconds before rinsing. After
decolourization, it is assumed that Gram-positive organisms keep the CVI complex while the
Gram-negative organisms become colourless. Furthermore, a counterstain of safranin is
added which is red in colour for another minute before rinsing and blotting dry. When viewed
under the microscope, the Gram-negative bacteria take the colour of safranin which is red
while the Gram-positive organisms remain purple because the pores are unable to absorb
the counterstain safranin. The safranin is therefore dehydrated by gram-positive bacteria and
remains purple meanwhile in gram-negatives, it can be absorbed because of their thin
peptidoglycan layer (Willey, et al., 2011).

Secondary screening is done to give answers to many different questions that may surface
during the final attempts to determine useful industrial microorganisms. Giant colony
technique is one of the simplest methods in secondary screening sometimes primary
screening, it is used to determine the antibiotic inhibition spectrum. The organism (antibiotic)
is streaked in a narrow band across the centre of the plates that have agar and incubated to
allow growth to occur. Strains of test organisms are streaked from the edges of the plates,
ensuring that it does not touch the central growth, then it is incubated. Measurements are
done on the distance over which the growth of each test organism has been inhibited by the
antibiotic. The zone is measured in millimetres. This technique is achieved by performing
experiments on agar plates or small bioreactors, sometimes both (Manwar, 2009). This type
of screening is mostly used in attempts to determine antibiotic properties from certain
organisms.

Aim: To investigate the production of antibiotic from actinomycetes isolated from soil and
plant and evaluate its antimicrobial activities
Objectives:

· To isolate, purify and characterize actinomycetes from soil and plant samples.
· To evaluate actinomycetes antimicrobial activity
Materials and Methods
Materials:

· Dilution bottles with 9ml sterile saline

· 100g of soil
· Leaves
· Bacteriological agar
· Petri dishes
· Calcium Carbonate, Potassium dihydrogen phosphate, Manganese sulphate, Zinc
sulphate, Ion sulphate and Sodium chloride.
· Gram stain reagents, slides, and microscope
· Inoculation equipment and Bunsen burner
Procedure:
Preparation of Isolation agar plates
Soil: 100g of garden soil was boiled for 5 minutes in 600ml distilled water to prepare soil
supernatant. The mixture was allowed to cool and settle then 500 ml was discarded to
another beaker and an agar containing 0.001g Calcium Carbonate, 0.25g Potassium
dihydrogen phosphate, 0.001g Manganese sulphate, 0.001g Zinc sulphate, 0.001g Ion
sulphate, 0.002g Sodium chloride and 1.5% agar was added. The mixture was then
boiled, poured into bottles and autoclave, therefore poured into plates, and stored in
refrigerator.
Plant: leaves were collected and boiled for 5 minutes in 600ml of distilled water then
allowed to cool and settle, therefore the mixture was discarded in 500ml beaker and the
same procedure as soil was followed but used plant sample supernatant.
Isolation of soil organisms
1g of soil was diluted to 10-7 in saline then spread plate the 10-3 to 10-7 dilutions. The
plates were incubated at 25°C for 3 days until growth was observed then colonies were
picked from the surface, Gramm stained and identified morphologically under
microscope. Once it was determined that the colony was Gram positive, it was then
streaked out on a soil isolation agar plate and a pure culture was transferred to a soil
isolation agar plate therefore incubated at 25°C.
Isolation of plant organisms
Two plant isolation agar plates were prepared then inoculated by pressing semi-dried
leave onto the surface of the agar and the plates were incubated for 2 days. Once
growth was observed, colonies were picked, and Gram stained therefore determined
morphology. A Gram-positive colony was determined therefore colonies were streaked
out on the plant isolation agar plate to obtain pure culture. Once a pure culture was
obtained, it was transferred to a plant agar plate and incubated at 25°C.
Part 2: Determination of antibiotic production by actinomycetes isolated from soil and
plant material by GIANT COLONY TECHNIQUE
Materials:

· Pure actinomycete cultures isolated from soil and plant material.

· Nutrient agar plates
· Test tubes containing broth.
· Test organisms for evaluating antibiotic production: Klebsiella, E. coli,
Staphylococcus, Proteus.
· Desiccator
· Chloroform
Procedure
Two nutrient agar plates were inoculated with a single line of purified Actinomycete
culture down the centre of the plate. One plate was for soil isolate and the other for plant
isolate. The plates were incubated for 2 days at 25° C until growth was observed then
the plates were placed open in a desiccator with a small container of chloroform for 20
minutes to kill the organisms. The plates were then inoculated with the test organisms
 then incubated at 37°C overnight. The distance of inhibition due to the production of an
antibiotic was measured the following day.

Results

Figure 1: Soil sample at different concentrations taken from serial dilution shows a number of
colonies on the surface of the soil agar spread plate. The colonies appeared in yellow and
brown colour.

Figure 2: Gram positive clustered cocci taken from pure culture of soil sample viewed under
1000x emission oil lens.
Figure 3: Giant colony of soil pure culture on a nutrient agar showing the different zone of

inhibition for the 4 test organisms. First line represents E. coli, 2nd represents C.cereus, 3rd
represents S.aureus and the last line represents K.pneumoniae.

Figure 4: showing the growth of mould and small brown and black colonies on the surface of
leaf agar plate after they have been semi dried and attached at the surface of the agar plate.
Figure 5: showing gram positive rods from leaves view under 1000x emission oil lens.

Figure 6: Giant colony of leaves showing different

zone of inhibition of the 4 test organisms. First line
represents E. coli, 2nd represents C.cereus, 3rd represents S.aureus and the last line
represents K.pneumoniae.

Table 1: shows the number of colonies grown on each of the spread plates for soil sample
taken from the garden.

Plate number Number of colonies on the

surface of the agar plates

10-3 23 colonies

10-4 1 colony

10-5 No growth (0 colonies)

10-6 No growth (0 colonies)

10-7 No growth(0 colonies)

Table 2: shows the results of each organism`s distance of inhibition for the production of an
antibiotic on both soil and leaf giant colony agar plates.

Organism Zone of inhibition for soil on Zone of inhibition for leaves

nutrient agar in (cm) on nutrient agar in (cm)

E. coli 0.2 and 0.3 Average (0.25) 0.4 and 0.3

S.cereus 0.2 and 0.2 Average (0.2) 0.1 and 0.1

S.aureus 0.4 and 0.3 Average (0.35) 0.2 and 0.3

K.pneumoniae 0.5 and 0.5 Average (0.5) 0

DISCUSSION
Actinomycetes are one of the most important microorganisms that produce a wide variety of
useful secondary metabolites, many of which have potent biological activities, including
many commercially important antibiotics and immunosuppressive compounds.

Antibiotics are the most important bioactive compounds for the treatment of infectious
diseases. But now, because of the emergencies of multi-drug resistant pathogens, there are
basic challenges for effective treatment of infectious diseases. Thus, due to the burden for
high frequency of multidrug resistant pathogens in the world, there has been increasing
interest for searching effective antibiotics from soil actinomycetes in diversified ecological
niches.

The spread plate was done from the serial dilution test tube 10 -3 to 10-7 and the results
obtained are shown in figure 1. The colonies appeared yellow and brown in colour and
mould was on the agar plate also. The number of colonies were easier to count on the first 2
plates that had a high soil concentration. Pure colonies from both soil and leaves culture
were picked using a loop and performed gram staining of which they both were gram
positive cocci clustered for soil and rods for the leaves as shown in figure 2 and 5. Table 2
shows the number of colonies on the spread plates from the serial dilution for the soil, since
they were less than 30 colonies of the first and second plate the colony forming units,
calculation could not be done.

Actinomycetes isolated and identified from soil and plant(leaves) samples were screened for
antibiotic production. The test bacteria used for primary screening were Escherichia coli (E.
coli), Staphylococcus aureus (S. aureus), S.cereus and K.pneumoniae . Nutrient agar plate
was streaked with isolated colonies of actinomycetes in the centre along the length and then,
fresh sub cultured test organisms were streaked perpendicular to the actinomycetes isolates.
Then the plates were incubated at 37°C for 24 hours. After incubation observation for zone
of inhibition was measured (cm) using a ruler and and the results were recorded as shown in
table 1. K.pneumoniae is the one that is more sensitive to the antibiotic produced by soil
actinomycetes in figure 3 while E.coli is the one that is more resistant to the antibiotic
produced by the leaves.

The survival of the microorganisms might be due to their adaptation and ability to produce
resistant structures such as colonies and spores. All the isolates were slow-growing, aerobic
and most of them produced pigments namely yellow and brown. The colonies were
extracted from actinomycete isolates and were purified and tested for antimicrobial growth
which showed that all extracted colonies inhibited growth of E. coli, Staphylococcus aureus,
S. cereus and K.pneumoniae which showed activity against test organisms among which 2
were active against Gram-negative and 2 against Gram-positive.

The reason for different sensitivity between gram-positive and Gram-negative bacteria could
be explained with respect to the morphological differences between these microorganisms.
All the isolates showed a zone of inhibition. The different activity might be due to the
differences in the substrate composition of the culture media, inoculum size and incubation
time. Different environmental constrains and growth conditions influence the growth and
diversity of actinomycetes, temperature being one of them. In the study, the optimum
temperature for the growth of actinomycetes was found to be in mesophilic range.

Conclusion

Actinomycetes are of great importance since they have the ability to contribute to human
well-being through the production of antimicrobial agents and other industrial products like
enzymes, drugs and natural pigments. Actinomycetes can be found in plants and soil
materials, they can even produce antibiotics. Soil and plant actinomycetes were found and
they produced antibiotics which inhibit E. coli and K.pneumoniae.

References
1. Abbas, S., Senthilkumar, R. and Arjunana, S. 2014. Isolation and characterization of
microorganisms producing novel antibiotics from soil sample. European journal of
experimental biology. 4(5): 149-155.
2. Gonzalez-Franco, C.A. and Rubles-Hernandez, R.Y. 2009. Actinomycetes as biological
control agents of phytopathogenic fungi. Tecnociencia Chihuahua. 3(2): 64-73.
3. Jeffrey, L.S. 2008. Isolation, characterisation and identification of Actinomycetes from
agriculture soils at Semongok, Sarawak. 7: 3697-3702.
4. Manwar, A. 2009. Industrial microbiology and screening. Industrial microbiology.
Available at: http://avmsidero.blogspot.co.za/2009/09/industrial-microbiology-and-
screening.html. Accessed: 07/04/2020.