This document summarizes a study analyzing the secondary metabolites produced by Pseudomonas fluorescens using gas chromatography-mass spectrometry (GC-MS) and evaluating its anti-fungal activity. GC-MS analysis identified 24 bioactive compounds in the bacterium's methanolic extract. Testing found that volatile compounds produced by P. fluorescens strongly suppressed the growth of the fungus Trichoderma horzianum. Several medicinal plants also demonstrated high antimicrobial activity against P. fluorescens and other microbes. The study concludes that P. fluorescens produces biologically active secondary metabolites with potential for development as new antimicrobial agents.

1. Analysis of Secondary Metabolites Released by Pseudomonas
fluorescens Using GC-MS Technique and Determination of Its
Anti-Fungal Activity
Rafid Hadi Hameed1
, Fatima Moeen Abbas2
, Imad Hadi Hameed3
1
Ministry of Public Health, Maysan Health Department, Mesan governorate, Iraq, 2
Department of Biology,
College of Science for Women, University of Babylon, Hillah City, Iraq, 3
Biomedical Science Department,
University of Babylon, College of Nursing, Hillah City, Iraq
ABSTRACT
Pseudomonas fluorescens is a common gram-negative, rod-shaped bacterium [1]. It belongs to the
Pseudomonas genus. The objectives of this study were analysis of the secondary metabolite products and
evaluation antimicrobial activity. Twenty four bioactive compounds were identified in the methanolic
extract of Pseudomonas fluorescens. GC-MS analysis of Pseudomonas fluorescens revealed the existence
of the cis-5,8,11,14,17-Eicosapentaenoic acid, 12,15-Octadecadiynoic acid, methyl ester, 7-epi-cis-
sesquisabinene hydrate, α-D-Glucopyranoside , O-α-D-glucopyranosyl, Acetamide, N-methyl-N-[4-
[2-acetoxymethyl-1- pyrrol, Acetamide, N-methyl-N-[4-[2-fluoromethyl-1- pyrrolid, Phen-1,4-diol ,
2,3-dimethyl-5-trifluoromethyl, Geranyl isovalerate, Quinazoline, 4-methyl, Pentetic acid, trans-13-
Octadecenoic acid, 9-Hexadecenoic acid, 13-Hexyloxacyclotridecan -2-one, 7-Methyl-Z-tetradecen-1- ol
acetate, cis-13-Eicosenoic acid, Didemin B, Hexadecanoic acid ,1-(hydroxymethyl) -1,2-ethaned, and Ethyl
iso –allocholate. Cassia angustifolia (Crude) was very highly active (6.007±0.20) mm against Pseudomonas
fluorescens. The results of anti-fungal activity produced by Pseudomonas fluorescens showed that the
volatile compounds were highly effective to suppress the growth of Trichoderma horzianum (5.019±0.18).
Pseudomonas fluorescens produce many important secondary metabolites with high biological activities.
Keywords: Anti-Microbial, Pseudomonas fluorescens, GC-MS, Secondary metabolites.
Corresponding author:
Imad Hadi Hameed
Biomedical Science Department, University of
Babylon, College of Nursing, Hillah city, Iraq;
Phone number: 009647716150716;
E-mail: imad_dna@yahoo.com
INTRODUCTION
P. fluorescens has multiple flagella. It has an
extremely versatile metabolism, and can be found in the
soil and in water. It is an obligate aerobe, but certain
strains are capable of using nitrate instead of oxygen as
a final electron acceptor during cellular respiration. P.
fluorescens is an unusual cause of disease in humans,
and usually affects patients with compromised immune
systems (e.g., patients on cancer treatment) 1-6
. P.
fluorescens isolates produce the secondary metabolite
2,4-diacetylphloroglucinol (2,4-DAPG), the compound
found to be responsible for antiphytopathogenic and
biocontrol properties in these strains. Several strains of P.
fluorescens, such as Pf-5 and JL3985, have developed a
natural resistance to ampicillin and streptomycin. These
antibiotics are regularly used in biological research as a
selective pressure tool to promote plasmid expression7-11
.
The aims of our research were analysis of the secondary
metabolite products of Pseudomonas fluorescens and
evaluation antimicrobial activity.
MATERIALS AND METHOD
Detection of secondary metabolites
Subcultures were obtained on the nutrient agar for 48
hrs. at 22Cº. Metabolites was separated from the liquid
cultureandevaporatedtodrynesswitharotaryevaporator
DOI Number: 10.5958/0976-5506.2018.00485.0

2. 446 Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5
at 45Cº. The residue was dissolved in 1 ml methanol,
filtered through a 0.2 μm syringe filter, and stored at 4Cº
for 24 h before being used for gas chromatography mass
spectrometry 12-23
. The identification of the components
was based on comparison of their mass spectra with those
of NIST mass spectral library as well as on comparison
of their retention indices either with those of authentic
compounds or with literature values24-35
.
Spectral analysis of bioactive natural chemical
compounds of Pseudomonas fluorescens using (GC/
MS)
Analysis was conducted using GC-MS (Agilent 789
A) equipped with a DB-5MS column (30 m×0.25 mm
i.d., 0.25 um film thickness, J&W Scientific, Folsom,
CA). The oven temperature was programmed as for the
previous analysis36-39
. Helium was used as the carrier gas
at the rate of 1.0 mL/min. Effluent of the GC column
was introduced directly into the source of the MS via a
transfer line (250 Cº)40-42
.
Determination of antibacterial and antifungal
activity
Five-millimeter diameter wells were cut from the
agar using a sterile cork-borer, and 25 μl of the plant
samples solutions were delivered into the wells. The
plates were incubated for 48 h at room temperature.
Antimicrobial activity was evaluated by measuring the
zone of inhibition against the test microorganisms43-47
.
The studied fungi, Microsporum canis, Aspergillus
flavus, Candida albicans, S. cerevisiae, Trichoderma
viride, Trichoderma horzianum, and Aspergillus terreus
were isolated and maintained in potato dextrose agar
slants. Spores were grown in a liquid culture of potato
dextrose broth (PDB) and incubated at 25ºC in a shaker
for 16 days at 130 rpm. The extraction was performed
by adding 25 ml methanol to 100 ml liquid culture in
an Erlenmeyer flask after the infiltration of the culture.
Methanol was used as solvent control. Amphotericin
B and fluconazole were used as reference antifungal
agent 48-50
. Results of the study were based on analysis
of variance (ANOVA) using Statistica Software. A
significance level of 0.05 was used for all statistical tests.
RESULTS AND DISCUSSION
Gas chromatography and mass spectroscopy
analysis of compounds was carried out in methanolic
extract of Pseudomonas fluorescens, shown in Table 1.
GC-MS analysis of Pseudomonas fluorescens revealed
the existence of the cis-5,8,11,14,17-Eicosapentaenoic
acid, 12,15-Octadecadiynoic acid , methyl ester, 7-epi-
cis-sesquisabinene hydrate, α-D-Glucopyranoside ,
O-α-D-glucopyranosyl, Acetamide , N-methyl-N-[4-[2-
acetoxymethyl-1-pyrrol,Acetamide, N-methyl-N-[4-[2-
fluoromethyl-1- pyrrolid, Phen-1,4-diol , 2,3-dimethyl-
5-trifluoromethyl, Geranyl isovalerate, Quinazoline ,
4-methyl, Pentetic acid, trans-13-Octadecenoic acid,
9-Hexadecenoic acid, 13-Hexyloxacyclotridecan
-2-one, 7-Methyl-Z-tetradecen-1- ol acetate, cis-
13-Eicosenoic acid, Didemin B, Hexadecanoic acid
,1-(hydroxymethyl) -1,2-ethaned, and Ethyl iso –
allocholate. The results of anti-fungal activity produced
by Pseudomonas fluorescens showed that the volatile
compounds were highly effective to suppress the
growth of Trichoderma horzianum (5.019±0.18)
Table 2. Pseudomonas fluorescens produce many
important secondary metabolites with high biological
activities. Based on the significance of employing
bioactive compounds in pharmacy to produce drugs
for the treatment of many diseases, the purification of
compounds produced by Pseudomonas fluorescens can
be useful. In agar well diffusion method the selected
medicinal plants Nerium olender (Alkaloids), Ricinus
communis (Alkaloids), Datura stramonium(Alkaloids),
Linum usitatissimum (Crude), Anastatica hierochuntica
(Crude), Cassia angustifolia (Crude), Althaea rosea
(Crude), Coriandrum sativum (Crude), Origanum
vulgare (Crude), Urtica dioica (Crude), Foeniculum
vulgare (Crude), and Ocimum basilicum (Crude)
were effective against Staphylococcus aureus, Table
3. Cassia angustifolia (Crude) was very highly active
(6.007±0.20) mm against Pseudomonas fluorescens.
Pseudomonas fluorescens was found to be sensitive to
all test medicinal plants and mostly comparable to the
standard reference antifungal drug Amphotericin B and
fluconazole to some extent.

3. Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5 447
Table 1. Major chemical compounds identified in methanolic extract of Pseudomonas fluorescens.
Molecular WeightRT (min)Phytochemical compoundSerial No.
302.224583.184cis-5,8,11,14,17-Eicosapentaenoic acid1.
290.224583.29312,15-Octadecadiynoic acid , methyl ester2.
222.1983653.6597-epi-cis-sesquisabinene hydrate3.
504.1690354.283α-D-Glucopyranoside , O-α-D-glucopyranosyl4.
266.1630424.609Acetamide , N-methyl-N-[4-[2-acetoxymethyl-1- pyrrol5.
226.1481424.672Acetamide , N-methyl-N-[4-[2-fluoromethyl-1- pyrrolid6.
206.0554645.295Phen-1,4-diol , 2,3-dimethyl-5-trifluoromethyl-7.
238.193286.016Geranyl isovalerate8.
144.0687487.899Quinazoline , 4-methyl-9.
393.1383448.322Pentetic acid10.
282.255888.551trans-13-Octadecenoic acid11.
254.2245810.7489-Hexadecenoic acid12.
282.2558812.22513-Hexyloxacyclotridecan -2-one13.
268.2402313.3187-Methyl-Z-tetradecen-1- ol acetate14.
310.2871813.375cis-13-Eicosenoic acid15.
308.1987613.6781,9-Dioxacyclohexadeca - 4,13-diene-2-10-dione , 716.
322.32356613.701Z-5-Methyl-6-heneicosen-11-one17.
1111.6416614.525Didemin B18.
568.50667614.719Hexadecanoic acid ,1-(hydroxymethyl) -1,2-ethaned19.
404.26750818.130Pregn-4-ene-3,20-dione , 17,21-dihydroxy-, bis(O-me20.
286.1932818.679Androst -5,7-dien-3-ol-17-one21.
488.24101819.743(22S)-21-Acetoxy-6α , 11β-dihydroxy-16α 17α-propy22.
436.31887421.843Ethyl iso -allocholate23.
422.19406722.7594H-Cyclopropa[5’,6’]bens[1’,2’:7,8]azuleno[5,6-b]ox24.
Table 2. Antifungal activity of Pseudomonas fluorescens metabolite products.
Microorganism Pseudomonas fluorescens products Fluconazol
Microsporum canis 3.116±0.16 ª 3.110±0.15
Aspergillus flavus 5.000±0.19 4.309±0.17
Candida albicans 4.702±0.17 2.873.±0.12
S. cerevisiae 3.005±0.16 2.000±0.11
Trichoderma viride 4.957±0.17 1.704±0.10
Trichoderma horzianum 5.019±0.18 4.005±0.19
Aspergillus terreus 4.951±0.16 3.251±0.17
ª The values ( average of triplicate) are diameter of zone of inhibition at 100 mg/mL crude extract and 30 μg/mL
of (Amphotericin B; Fluconazol and Miconazole nitrate).

4. 448 Indian Journal of Public Health Research & Development, May 2018, Vol. 9, No. 5
Table 3. Zone of inhibition (mm) of test different
bioactive compounds and standard antibiotics of
medicinal plants to Pseudomonas fluorescens.
S.
No.
Plant
Zone of
inhibition
(mm)
1. Nerium olender (Alkaloids) 3.779±0.17
2. Ricinus communis (Alkaloids) 2.639±0.15
3. Datura stramonium(Alkaloids) 3.990±0.16
4. Linum usitatissimum (Crude) 4.815±0.18
5. Anastatica hierochuntica (Crude) 5.941±0.19
6. Cassia angustifolia (Crude) 6.007±0.20
7. Althaea rosea (Crude) 5.000±0.18
8. Coriandrum sativum (Crude) 5.943±0.19
9. Origanum vulgare (Crude) 5.799±0.18
10. Urtica dioica (Crude) 4.228±0.16
11. Foeniculum vulgare (Crude) 3.000±0.14
12. Ocimum basilicum (Crude) 5.716±0.17
13. Control 0.00
CONCLUSION
Twenty four bioactive chemical constituents
have been identified from methanolic extract of the
Pseudomonas fluorescens by GC-MS. In vitro antifungal
and antibacterial evaluation of secondary metabolite
products of Pseudomonas fluorescens forms a primary
platform for further phytochemical and pharmacological
investigation for the development of new potential
antimicrobial compounds.
Financial disclosure: There is no financial
disclosure.
Conflict of Interest: None to declare.
Ethical Clearance: All experimental protocols
were approved under the Department of Biology, College
of Science, Hillah city, Iraq and all experiments were
carried out in accordance with approved guidelines.
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