Isolation and Identification of antibiotic produced by Penicillium chrysogenum isolated from different environments in Nasseriya city soils
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2017-11-18
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Abstract
The present study includes the isolation and identification of some
mycoflora from 40 soil samples in six places ( Remnants of fat-born,
parks, edges of the river, animal wastes, sewage and rubbish) during
October 2015–January 2016 in Nasseriya City, Iraq. According to
different environmental factors, the isolated genera were Aspergillus,
Penicillium, Mucor, Rhizopus, Cladosporium, Sepedonium
chrysospermum, Alternaria chlamydospora, Bipolaris, Chrysosporium,
Candida albicans, Rhododendron flavum, Humicola, Geotrichum
candidum, Fusarium and Acremonium. Three isolation methods were
used. Dilution method, direct plate method, alcohol and heat treatment
technique using the cultural media viz. Potato Dextrose Agar ( PDA),
Sabouraud Dextrose Agar (SDA) and Potato Carrots Agar (PCA).
Aspergillus represented the highest fungal isolates which represent 62
(37.12%). followed by Penicillium with 47 (28.14%), Mucor 22
(13.16%), Rhizopus 15 (8.98%), Cladosporium 6 (3.59%), Sepedonium
chrysospermum and Alternaria chlamydospora, 3 (1.80%), Biopolaris,
Chrysosporium and Candida albicans with 2 (1.20%), and finally
Rhododendron flavum, Humicola, Acremonium, Fusarium and
Geotrichum candidum recorded the lowest fungal isolation with one
isolate for each (0.60%). The study was aimed to isolation of Penicillium
from soil and assay its antibacterial activity. The results showed that
dilution method gave a best fungal growth in comparison with direct
plate method and alcohol and heat treatment technique in 25 ºC and pH =
6. PDA appeared as an optimum medium for isolation in comparison with
other media such as SDA and PCA. The preliminary results showed that
Penicillium sp. exerted antibacterial activity against Gram positive and
Gram negative bacteria; Therefore, this fungus was used for production
of antibiotic on Potato Dextrose Broth (PDB) medium. The optimum
conditions were obtained at pH = 6, incubation temperature of 25 ºC and
shaking rate of 180 rpm for 7 days to fermentation. Ethyl acetate was a
good organic solvent to extraction of antibiotic which produced (3g/L) as
white to creamy crystals. The characterization of the antibiotic product
after extraction and purified by chemical methods included Thin Layer
Chromatography (TLC) test, Nuclear Magnetic Resonance (NMR)
Spectra and Mass Spectra. The Minimum Inhibitory Concentration (MIC)
for clinical bacterial species was 1-10mg/ml.
The PCR method used in this study utilized Internal transcribed spacer
(ITS1-4) as a primer for identification of isolates. Two PCR products of
the targeted gene of Penicillium chrysogenum isolates which locally
named as (Penicillium chrysogenum F1 and Penicillium chrysogenum F2)
were selected and subjected to partial DNA sequencing for the ITS gene
to follow up the possible molecular relationship between these local
isolates and what recorded globally in Genbank.