Industrial Microbiology

Industrial Microbiology-2nd ppt

Prof. Hyang Burm Lee
Agricultural Chemistry,
College of Agriculture and Life Sciences,
Chonnam National University,
1
Naila Khan Bangash
2
Contents:
• Endophytes
• Fungal diversity
• Aim of study
• Results and Discussions
• Silver nanoparticles
• Antibacterial activity of nanoparticles
• Cytotoxicity of nanoparticles
• Conclusions

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 Endophytes
• Endophytes are the microbes that colonize living, internal tissues of plants with-
out causing any immediate, overt negative effects.

Significance of endophytic Diversity

- Endophytic mycobiome forms a significant part of the microbial diversity.
- Endophytes comprise a large hidden component of fungal biodiversity.
- Taxol (Plaxitaxel): Produced by Taxomyces andreanae, endophytic fungus from Taxus brevifo-
lia
- Unexplored or untapped source of novel bioactive metabolites.
- Diverse biological activity due their unpresedented structural diversity (antibacterial, antifun-
gal, antitumoral, antiviral, antioxidant, imunossupressor, insecticide) (Strobel et al., 2004)
 Fungal diversity

Endophytic fungi inhabit almost all variety of plant species. The diversity of fungal endo-
phytes mainly depends on the type of plant species and geographical area. Plants with eth-
nobotanical history form important criteria towards screening of endophytes which may lead
to isolate endophyte with potent activity and few of the scientific reports suggests that
plants growing in rich biodiversity lodge diverse group of endophytes with activity.
 Antimicrobial
agents

Plant growth
Anti-diabetic
Promoters

Fungal en-
dophytes
Agro
Anti cancer-
Important ous
metabolites

Anti inflam-
Anti-diabetic
matory

Plant-associated microorganisms, especially endophytic fungi are largely underexplored in the

discovery of natural products (Strobel and Daisy, 2003; Gunatilaka et al., 2006)
 Medicinal plants selected for the isolation
of endophytic fungi

Figure: Habit of Mirabilis jalapa Linn.
Botanical Name: Mirabilis jalapa Linn.
Family: Nyctaginaceae (Bougainvillea family)
Common name: Four O’ clock plant, Beauty-of-
the-night, Marvel of Peru, Gulamaji (Local Na-
tive Name)
Figure: Habit of Ficus pumila Linn.
Botanical Name: Ficus pumila Linn.
Family: Moraceae (Mulberry family)
Common name: Creeping fig
 Aim of the present investigation
Pla • Plant materials of Ficus pumila and Mirabilis jalapa were collected in
nt
col-
three different season i.e., summer, monsoon and winter.
lec-
tion
Iso-
la- • Isolation of endophytic fungi was carried out.
tion
of
en-
do-
phyt
es • Identification of endophytic fungi was carried out based on the molecu-
Ide
nti lar, macroscopic and microscopic observations.
fi-
cat
ion
• To assess and quantify endophytic fungal diversity between two host
plants following diversity indices were used
Dat
a • Colonization rate (CR)
anal • Isolation rate (IR)
yses • Colonization frequency (CF)
Fig: Overview of the procedure of isolation, cultivation and identification of endophyte fungi
 Results 1200 plant
segments

238
(19.83%)

23 Taxa, comprising

3 Ascomycetes genera: Chaetomium sp., Sporomia sp. and Xylaria sp. (10.92%),
5 Coelomycetes genera: Colletotrichum sp., Pestalotiopsis sp., Phoma sp., Phomopsis sp. and Phyllosticta sp.
(18.06%),
11 Hyphomycetes genera: Acremomium sp., Alternaria sp., Aspergillus sp., Cladosporium sp., Curvularia
sp., Drechslera sp., Fusarium sp., Myrothecium sp., Nigrospora sp., Penicillium sp. and Trichoderma sp.
(55.46%)
2 Zygomycetes genera: Mucor sp. and Rhizopus sp. (2.94%),
2 Morphospecies of Mycelia sterilia (8.4%).
 Colonization and Isolation rates

 99 segments (50.51%) of the stem and 97 segments of (49.48%)

leaf were colonized with endophytic fungi from both the plants.
 Colonization rates (%) of endophytic fungi for both the tissues
were found to be relatively homogeneous (14.33% and 14.0%)
and (18.66% and 18.33%) for Mirabilis jalapa and Ficus
pumila.
 Isolation rates of endophytic fungi in leaves of both plants were
relative high when compared to stem in both plants.
 Colonization frequency (CF)
Aspergillus flavus and Xylaria sp., are dominant endophytic fungal genera in leaf while in stem species of
Fusarium and Pestalotiopsis were dominant in stem of Mirabilis jalapa and Ficus pumila respectively.

Dominance and species richness

 Despite the similar patterns of species accumulation, endophytes segregated with varying degree of colonization

during three sampling season among different tissues of the two hosts.
 Overall diversity indices values for foliar endophytes of Ficus pumila was relatively high when compared to leaf

and stem tissues of Mirabilis jalapa.

 But species richness was higher in stems of Mirabilis jalapa when compared with leaves and stems of Ficus pumila

due to isolation 19 different endophytic fungal taxa.

Data Analysis

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Cultural , Morphological and Microscopic Characteristics of
Major endophytic fungal genera isolated in the present study

Cladosporium sp. Colletotrichum sp. Curvularia sp. 1

Curvuilaria sp. 2 Dreschlera sp. Fusarium sp. 1

 A. flavus A.niger Acermonium sp.

Alternaria sp. Chaetomium sp. 1 Chaetomium sp. 2

F. oxysporum Fusarium sp. 2 Morphospecies sp. 1

Morphospecies sp. 2 Morphospecies sp. 3 Morphospecies sp. 4

F. oxysporum Fusarium sp. 2 Morphospecies sp. 1

Morphospecies sp. 2 Morphospecies sp. 3 Morphospecies sp. 4

Pestalotiopsis sp.2 Phoma sp Phomopsis sp 1

Phomopsis sp 2 Trichoderma sp
Table 1. Isolation and colonization and rate of endophytic fungi of Mirabilis jalapa Linn.
Stem Leaf Total
No. of segments 300 300 600
No. of segments yielding fungi 21+12+10=43 16+19+07=42 85
No. of isolates 26+14+10=50 20+24+07=51 101
Isolation rate 0.16 0.17 0.16
Colonisation rate (%) 14.33 14 14.16

Table 2. Isolation and colonization rate of endophytic fungi of Ficus pumila Linn.
Stem Leaf Total
No. of segments 300 300 600
No. of segments yielding fungi 15+21+20 =56 18+19+18 =55 111
No. of isolates 16+23 +23 =62 26 +25 +24=75 137
Isolation rate 0.20 0.25 0.22
Colonization rate (%) 18.66 18.33 0.18
Table 3. Diversity of endophytic fungi isolated from stem and leaf of Mirabilis jalapa Linn. during three different season (2010-2011)
Class Endophyte Stem CF (%) Leaf CF (%)
Ascomycetes Chaetomium sp.1 2 0.66 1 0.33
Chaetomium sp.2 4 1.3 - -
Sporormia sp. - - 1 0.33
Coelomycetes Phoma sp. 3 1.0 - -
Phomopsis sp.1 8 2.6 - -
Phomopsis sp.2 3 1.0 - -
Hyphomycetes Acremonium sp. - - 1 0.33
Alternaria alternata - - 2 0.66
Aspergillus flavus - - 7 2.33
Aspergillus niger 1 0.33 5 1.66
Aspergillus sp.1 1 0.33 2 0.66
Aspergillus sp.2 - - 1 0.33
C. Cladosporioides - - 2 0.66
C. herbarum 1 0.33 1 0.33
Cladosporium sp. - - 2 0.66
Curvularia sp.1 1 0.33 3 1.0
Curvularia sp.2 1 0.33 - -
Drechslera sp. 1 0.33 3 1.0
Fusarium sp.1 8 2.6 - -
Fusarium sp.2 4 1.3 - -
Fusarium oxysporum - - 2 0.66
Penicillium sp. - - 2 0.66
Penicillium chrysogenum 1 0.33 3 1.0
Trichoderma sp.1 6 2.0 - -
Trichoderma sp.2 2 0.66 - -
Zygomycetes Mucor sp.1 1 0.33 - -
Rhizopus sp.1 1 0.33 2 0.66
Mycelia sterilia Morphospecies-1 1 0.33 5 1.6
Morphospecies-2 - - 6 2.0
 Table 4. Diversity of endophytic fungi isolated from stem and leaf tissue of Ficus pumila Linn.
Class Endophyte Stem CF (%) Leaf CF (%)
Ascomycetes Chaetomium sp. 01 0.33 05 1.66
Sporormia sp. - - 01 0.33
Xylaria sp. - - 11 3.66
Coelomycetes Colletotrichum sp. 02 0.66 03 1.00
Pestalotiopsis sp.1 07 2.33 - -
Pestalotiopsis sp.2 07 2.33 - -
Phoma sp. 06 2.00 - -
Phomopsis sp.1 06 2.00 - -
Phomopsis sp.2 06 2.00 - -
Phyllosticta sp. - - 02 0.66
Hyphomycetes Acremonium sp. - - 03 1.00
Alternaria alternata - - 06 2.00
Alternaria sp. - - 01 0.33
Aspergillus flavus - - 04 1.33
Aspergillus niger - - 04 1.33
Aspergillus sp. - - 06 2.00
Cladosporium sp. - - 05 1.66
Curvularia sp. - - 07 2.33
Drechslera sp. - - 06 2.00
Fusarium sp. 07 2.33
Myrothecium sp. 06 2.0 - -
Nigrospora sp. 04 1.3 - -
Penicillium sp.1 - - 04 1.33
Trichoderma sp. 06 2.0 - -
Zygomycetes Rhizopus sp. 02 0.66 01 0.33
Mycelia Sterilia Morphospecies-1 02 0.66 04 1.33
Morphospecies-2 - - 02 0.66
Table 5. Seasonal distribution of endophytic fungi in different tissues of Mirabilis jalapa Linn.
during three different season (2010-2011).

Endophytes Tissue of the Summer Monsoon Winter

host plant No. of No. of No. of
endophytes endophytes endophytes
Ascomycetes Stem 0 3 3
Leaf 2 0 0
Coelomycetes Stem 3 9 2
Leaf 0 0 0
Hyphomycetes Stem 7 11 9
Leaf 3 16 17
Zygomycetes Stem 0 2 0
Leaf 0 0 2
Mycelia sterilia Stem 0 1 0
Leaf 2 4 5
Total 17 46 38
Table 7: Seasonal distribution of endophytic fungi in different tissues of Ficus pumila Linn.
during three different season (2010-2011)

Endophytes Tissue of the Summer Monsoon Winter

host plant No. of No. of No. of
endophytes endophytes endophytes
Ascomycetes Stem 00 01 0
Leaf 06 6 5
Coelomycetes Stem 10 12 12
Leaf 03 1 1
Hyphomycetes Stem 04 8 13
Leaf 17 16 11
Zygomycetes Stem 00 02 0
Leaf 00 0 1
Mycelia sterilia Stem 02 00 0
Leaf 00 02 4
Total 42 48 47
Silver nanopartices

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Antibacterial activity of
nanoparticles

Bacterial cell

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Cytotoxicity of nanoparticles

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 Conclusions

 Endophytic fungi are more efficient in production of

nanoparticles.
 Biocompatible, stable, reproducible, and potent.
 Significant antimicrobial activity.
 Significant cytotoxic antivity.
 Nanoparticles disturb the bacterial pathways.
 High content is toxic to eukaryotic cells.

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Contents:
• Abstract
• Introduction
• Aim of study
• Materials and Methods
• Results and Discussion
• Conclusions
• Future perspectives

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 Abstract

• The endophytic fungal populations of Citrus jambhiri.

• Sources of novel bioactive molecules.
• Solid state fermentation on rice medium.
• Metabolites extraction using ethyl acetate.
• Screening for antimicrobial activity.
• Bioactive compounds detection using HPLC analysis.
• Antibacterial activity only against Staphylococcus aureus.
• Three bioactive compounds: protocathechuic acid, indole-3-
acetic acid, and acropyrone.

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 Introduction

• Citrus species- highly nutritious plants.

• Rutaceae have high amount of active compounds.
• Vitamin C, carotenoids (β-carotene), flavonoids, essential oils,
alkaloids, high quality soluble fibre, minerals, vitamin-B com-
plex and related nutrients such as thiamine, riboflavin, nico-
tinic acid/niacin, pantothenic acid, pyridoxine, folic acid, biotin,
choline, and inositol.
• Antibacterial, antiviral, anti-allergic, anticarcinogenic and anti-
fungal activities are reported.

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 Aim

• Few studies on endophytes of citrus plants.

• Citrus jambhiri have medicinal importance.
• Several endophytic bacterial and fungal population.
• To isolate endophytic fungi.
• To isolate biologically important molecules.
• To identify bioactive metabolites.

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 Materials and Methods

Isolation of
Extraction of
endophytic Fermentation
metabolites
fungus

HPLC analy-
Antibacterial Antifungal as-
assay say sis

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 Materials and Methods

Isolation of endophytic fungus

Surface ster-
ilization us-
ing 2% Leaf frag- Monitoring
Citrus jambiri Incubated at Sub-cultured
NaOHCl, ments put of fungal
leaves 28˚C on MEA
70% ethanol, into MEA growths
rinse in ster-
ile water

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 Materials and Methods

Solid state fermentation

Autoclaved rice medium (1:1) in 3 mm diameter agar blocks of Incubated at 28˚C for 21
Erlenmeyer flask fungi days

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 Materials and Methods

Metabolites extraction

Concentration under vac-

Secondary metaboliates in the fer-
Extraction with ethyl acetate cum at 40˚C using rotaory
mentation medium
evaporator

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 Materials and Methods

Antimicrobial assay

1 mg/mL of ex-
tract tested
against Staphy-
Also tested
lococaureus, Measured In-
Antibacterial and Agar well diffu- agaist As-
Salmonella ty- hibition zone
antifungal activity sion method pergillus fumi-
phi, Bacillus diameters
gatus
subtilis, E. coli,
Candida albi-
cans

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Antibacterial activity

Positive controls: Gentamicin and Ke-

toconazole
Negative control: DMSO

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 Materials and Methods

Antimicrobial assay

1 mg/mL of ex-
tract tested
against Staphy-
Also tested
lococaureus, Measured In-
Antibacterial and Agar well diffu- agaist As-
Salmonella ty- hibition zone
antifungal activity sion method pergillus fumi-
phi, Bacillus diameters
gatus
subtilis, E. coli,
Candida albi-
cans

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 Materials and Methods

HPLC Analysis

100 μL dis-
solved
Crude fungal
sample
extract (2 Vial put in Absorption
Cen- trans-
mg) reconsti- HPLC Detection peak analy-
Sonication trifuged at ferred to
tuted with 2 machine found at sis by com-
for 10 min 3000 rpm 500 μL 235 nm pairing eith
mL of HPLC for analy-
for 5 min HPLC database
grade sis
grade
methanol
methanol
(in a vial)

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 Results and Discussion

Table 1: Results of the antimicrobial evaluation of the fungal extract showing

the inhibition zone diameters (IZD) (mm) produced against test organisms.

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 Results and Discussion

• HPLC Chromatogram:

Figure 1: HPLC chromatogram of the endophytic fungal extract showing the detected com-
pounds - (A) Protocathechuic acid, (B) Indole-3-acetic acid and (C) Acropyrone.

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 Results and Discussion
• UV Spectra and Chemical Structure of Detected Bioactive
Compound:

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 Results and Discussion
• UV Spectra and Chemical Structure of Detected Bioactive
Compound:

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 Results and Discussion
• UV Spectra and Chemical Structure of Detected Bioactive
Compound:

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 Conclusions

• Endophytic fungi associated with C. jambhiri could be a poten-

tial source of novel compounds.
• Pharmaceutical and industrial applications.
• Posees biological activities that are antimicrobial, cytotoxic,
anti-inflammatory or antioxidant.

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 Future perspectives

• Further study to identify the endophytic fungi that have diverse

industrial application.
• A better understanding of endophytic fungi and their implemen-
tation in nanoparticles production.
• To explore antimicrobial activity by nanoparticles.
• To minimize their toxicity and maximize the biocompatibility
and effectiveness.

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