Isolation and Characterization of Antibiotic Production from Soil Isolates by Fermentation

Chandrashekhara S²*, BK Nanjwade¹, PS Goudanavar²,FV Manvi¹ Shamrez Ali M¹

¹KLES’s College of Pharmaceutical Sciences, K.L.E. University, Nehru Nagar, Belgaum, Karnataka, India.

²KLES’s College of Pharmaceutical Sciences, Nipani, Karnataka, India,

ABSTRACT

In screening of new antibiotics, several actinomycetes were isolated from soil samples. Crowded plate technique was used for the isolation of actinomycetes. The morphological and cultural characterization of A-4 strain was performed. In medium formulation study for A-4 and A-4 mutant, various carbon and nitrogen sources were tested for maximum antibiotic production using zone of inhibition and packed cell volume (%) as parameters. Various fermentation conditions like pH, temperature and DO₂ were also optimized for the maximal production of antibiotic from both A-4 and A-4 mutant. All medium formulation as well as bioprocess parameters for A-4 and A-4 mutant strains was compared. Some actinomycetes strains, showed promising antimicrobial scores against different strains of bacteria and fungi. From the six strains selected, one strain designated as A-4 showed maximum antimicrobial property against gram positive and gram negative strains as well as various fungi. Morphological and cultural studies showed that A-4 is belongs to actinomycete genus.. The strain A-4 and A-4 mutant was found to be having better antimicrobial activity in comparison with other soil isolates of actinomycetes.

KEYWORDS: Actinomycete, Antibiotic, Crowded plate technique, Zone of Inhibition, Fermentation.

INTRODUCTION

Antibiotics are the best known products of actinomycete. Over 5000 antibiotics have been identified from the culture of gram positive, gram negative organisms and filamentous fungi, but only 100 antibiotics have been commercially used to treat human, animal and plant disease. The genus Streptomycete is responsible for the formation of more than 60% of known antibiotics. While further 15% are made by number of related Actinomycete, Micromonospora, Actinomadura, Streptoverticillium and Thermoactinomycetes¹.

Antibiotics, because of their industrial importance, are the best known products of actinomycetes. Streptomycete is responsible for the formation of more than 60% of known antibiotic while further 15% are made by number of related actinomycete like Micromonospora, Actinomadura, Stretoverticillium and Thermoactinomycetes².

The actinomycetes produce an enormous variety of bioactive molecules e.g. antimicrobial compounds. One of the first antibiotics used was Streptomycin produced by Streptomycin griseus. The last 55 years have seen the discovery of more than 12,000 antibiotics. The actinomycetes produce about 70% of these, and the remaining 30% are products of filamentous fungi and non-actinomycete bacteria. Most of the bioactive compounds from actinomycete sort into several major structural classes like amino glycosides (e.g. Streptomycin and Kanamycin), ansamycins (Rifampin), anthracyclines (doxorubicin), b-lactum (cephalosporins), macrolides (erythromycin), and tetracyclines.

The present study is an effort in the direction of isolating an antibiotic capable of acting on clinical resistant strains of infectious organisms. Initially clinical resistant organisms were collected. Soil samples were screened for the presence of antibiotic producers. Many antibiotic producers were detected and a few were selected based on spectrum of activity. Soil isolate coded as A-4 was selected for further study.The antimicrobial activity was determined.

MATERIALS AND METHODS:

MATERIALS:

Dextrose IP, NaCl, Soyabean meal, n-Butanol, Silicon oil, Dimethyl sulphoxide (DMSO), n-Hexane, Tryptone, Lactose, Maltose, Fructose, Yeast extract powder, Nutrient agar, Agar powder ISP 2 to 7 media, Actinomycetes agar, Bennett’s agar, Nutrient broth, Sucrose, Silica gel 60-120 mesh, Chloroform, Ethyl acetate, Starch (NICE),Magnesium sulphate, Potassium dihydrogen phosphate, Methanol, Silica gel TLC grade, Peptone, Skimmed milk and Glucose. All chemicals were of analytical/Pharmacopoeia grade from commercial suppliers and were used as received without further purifications.

Methods:

1. ISOLATION and Characterization of Soil Isolates:

a) Collection and Screening of soil samples for actinomycetes:

The soil samples were collected from places, in and around Belgaum, Karnataka, India. The soil samples were dried separately at 45°C for 1 hr in hot air oven. Then they were cooled to room temperature. 1 gm of each soil sample was added to a conical flask containing 100 ml of sterile water and few drops of Tween-80 solution. All flasks were shaken for 30 minutes in orbital shaker incubator at 27°C. These flasks were considered as stock cultures. Soil samples for actinomycetes, which are capable of producing antibiotic, were screened by crowded plate technique. The actinomycete colonies isolated from the crowded plate were selected for the further studies which were named as A1, A1.5, A2, and A2.5 …..A9. ^{3, 4 5}

b) Preliminary screening of crude antibiotic produced:

Agar Streak Method:-

The microbial sensitivity of the soil isolates were analyzed by ‘Agar streak method’. The length of inhibition given against each test organism was observed. The isolated actinomycetes were screened against microorganisms such as Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Aspergillus niger and Candida albicans ^{6, 7, 8}.

c) Cultural and Microscopical characterization:

Morphological and cultural characters of the selected actinomycetes strains were studied by inoculating the selected strain into sterile International Streptomycetes Project (ISP) media. Morphological characters such as colony characteristics, type of areal hyphae, growth of vegetative hyphae, fragmentation pattern and spore formation were observed. Microscopical characterization was done by Gram staining method. ⁹

2. FERMENTATION PROCESS:

Down stream process for fermentation:

The fermentation was carried out for 4 days, keeping all the optimized fermentation parameters constant. After fermentation, the medium was centrifuged at 10,000 rpm for half an hour at 4°C to remove the biomass and cell debris. The supernatant was separated. The cell pellet of biomass was triturated with sterile sand to disrupt the cell wall. It was then washed with tris buffer (pH 7.4) filtered and centrifuged. The supernatant which contained all intracellular components was separated out. Both supernatants were checked for its antimicrobial activity. The supernatant (No.1) showed antimicrobial activity. This indicates that antibiotics which can produce by strain A-4 is present extracellularly. ^10-12

Following fermentation parameters were found to be ideal for maximum antibiotic productivity using A-4 strain of actinomycete.

1) Temperature: 28°C 2) pH: 7.0 3)DO₂concentration: 60% 4)Harvesting time (Fermentation duration): 96 hrs 5) Inoculum concentration: 10%v/v

3. DETERMINATION OF ANTIMICROBIAL ACTIVITY:

It was determined by cup plate technique. The test Bacteria were grown on nutrient agar, where as fungi were grown on Sabouraud dextrose agar medium (Himedia Labs, Mumbai).The extracts were dissolved in ethyl acetate and 50 µL of the samples were placed in to the corresponding cups. Growth inhibition was measured after 24h incubation at 37◦C for bacteria and after 72 h incubation at 27◦ C for fungi. The antimicrobial activity was estimated by measuring the inhibition zone diameters. ^{8, 12}

RESULTS AND DISCUSSION:

1. Isolation and CHARACTERIZATION of Soil Samples:

a) Screening of Soil Samples:

In the course of screening for novel antimicrobial substances (antibiotics) from soil samples, antibiotic-producing actinomycete cultures were recorded from soil samples taken in Belgaum, Karnataka. Actinomycetes have provided many important bioactive compounds of high commercial value and continue to be routinely screened for new bioactive substances. In the present study, about eighteen actinomycetes were isolated from soil samples, with isolation media as soyabean-casein digest medium (SBCD), actinomycetes isolation agar (AIA) and starch-casein agar, having pinpoint colonies with zone of inhibition, cultured by crowded plate technique. Isolation of actinomycetes from the isolation media was carried out by multiple streak method in actinomycetes agar media to obtain the pure cultures of eighteen actinomycetes strains. Each purified strain was preserved in actinomycetes agar slants at 4°c.

The soil samples were collected aseptically and processed within two hours after the heat treatment which inhibits growth of unwanted bacteria and fungi. Addition of CaCO₃ and heat treatment helped in the making up of actinomycetes populations in the soil.

The presence of relatively large populations of actinomycetes in soil samples of Belgaum indicates that it is an eminently suitable ecosystem which helps to isolate actinomycetes from screening programs. The details of collection of soil samples are shown in table 1. (Plate No.1)

Table No.1: Collection of Soil Samples and Number of Actinomycetes in Isolation Plates

Sr. No.

Dilution of soil samples (10^-5)

Heat treatment (45°C, 1 hr)

No. of actinomycetes colonies on isolation media

Nature of soil sample

pH of Soil

SBCD

AIA

SCA

10-4

10-3

10-4

10-3

10-4

10-5

Water logged mud

Loamy

Sandy

Mud

Red-dry soil

red-dry soil

Black-dry soil

7.5

6.3

7.0

8.0

7.8

5.3

5.7

5.4

NA: Not applied, AP: Applied, SBCD – Soyabean casein digest medium AIA – Actinomycetes isolation agar, SCA – Starch casein agar

Table No.2: Sensitivity of different microorganisms towards the soil isolates by agar

Soil isolates

B. subtilis

S. aureus

E. coli

K. pneumoniae

P. aeruginosa

A. niger

A. terreus

C. albicans

+++

+++ = Better inhibition, ++ = Good inhibition, + = Moderate inhibition, - = No inhibition

Table No.3: Morphological and cultural characterization of the strain A-4

Sr. No.	Medium Used	A4
1.	Tryptone-yeast extract broth (TSP-1)	Growth occurs by the pellicle formation.
2.	ISP-2	Creamish white colored colonies with clear zone around it were observed.
3.	(Oatmeal agar) ISP-3	Slight black – creamish color thick colonies; no aerial mycelium formation was observed.
4.	Inorganic salt-starch agar (ISP-4)	Blackish-brown colored thick colonies with waxy margin and convex surface was observed.
5.	Glycerol asparagines agar base (ISP-5)	Whitish colored thin colonies striated surface; with less aerial mycelium and filamentous growth was observed.
6.	Peptone yeast extract iron agar (ISP-6)	Thin transparent colonies with black colored soluble pigments were seen. No filamentous growth was seen.
7.	Tyrosine agar base (ISP-7)	Cream colored, lobe shape, convex surface, little mycelium growth was observed.
8.	Carbon utilization agar (ISP-9)	Thin yellowish golden colored colonies with little mycelium growth were observed.

Table No.4: Optimization of duration of fermentation for the maximum growth and antibiotic production

Sr. No.

Time in hrs

A-4

A-4 mutants

PCV (%)

Zone of inhibition (diameter in cm)

PCV (%)

Zone of inhibition (diameter in cm)

0.1

0.2

0.25

0.4

0.7

0.9

1.5

2.0

2.3

2.2

2.4

2.5

2.7

0.02

0.1

0.3

0.4

0.6

0.9

1.5

3.3

2.1

2.4

2.7

2.8

3.2

3.6

Well diameter = 0.9 cm, Organism used = Klebsiella pneumoniae

b) Test for Microbial Sensitivity:

The isolated strains of actinomycetes were tested for microbial sensitivity against five bacterial strains and three fungal strains by agar streak method (Table 2).

Out of eighteen actinomycetes screened, six strains namely A1, A2, A3, A4, A5 and A6 showed significant antimicrobial activity against both gram-positive and gram-negative organisms. However, A4 showed a very broad spectrum with higher scores than all other strains.

c) Morphological and Cultural characterization of A-4 strains:

Morphological characteristics of A-4 strains in different ISP media, showed the filamentous growth in ISP-5 and ISP-7 media and the pigmentation was seen in ISP-6 medium. The morphological characters of strain A-4 were also studied by microscopical observation after Gram-staining method. The observations revealed that A-4 strain is gram positive and rod shaped microorganism. The microscopical characteristics were observed under 10x and oil-immersion (100x). Morphological characters such as colony characteristics, type of areal hyphae, growth of vegetative hyphae, fragmentation pattern and spore formation are shown in Table 3.

By studying the morphological and cultural characteristics, it is observed that strain A-4 is belong to the genus actinomycetes.

2. FERMENTATION PROCESS:

The fermentation batch processed containing optimized medium formulation and fermentation conditions, was studied to determine the maximum duration of fermentation and results are shown in the Table No.4.

3. Determination of Antimicrobial Activity:

The antimicrobial activity in terms of minimum inhibitory concentration (MIC) of an isolated compound from A-4 and A-4 mutant fermentation broth was studied. The antibiotic showed broad spectrum of activity against gram positive and gram negative organisms and the MIC was found to be in the range of 100-125 mg/ml.The compound also possess significant antifungal activity against fungal strains tested, and the MIC was found to be 125 mg/ml. (Table No.5).

Table No.5: Determination of MIC for an isolated antimicrobial compound against Bacteria and Fungi

Test microorganisms (Bacteria)

MIC of isolated compound in mg/ml

A-4

A-4 mutant

A. Gram positive organisms

1. Staphylococcus aureus

2. Bacillus subtilis

125

100

B. Gram negative organisms

1. Escherichia coli

2. Pseudomonas aeruginosa

3. Klebsiella pneumoniae

125

100

62.5

Test microorganisms (Fungi)

1. Candida albicans

2. Saccharomyces cerviciae

125

62.5

4. IDENTIFICATION OF COMPOUND:

The structure of the isolated compound was characterized using I.R. Spectroscopy. Data and Functional groups are shown in Table No.6.

Table No.6: IR spectroscopic data and their functional groups

Sr. No.

Wave number

Functional groups

3424

1414

1677

1099

3147

Secondary amines

Alkyl groups

Aldehydic groups

Alcoholic groups

Aromatic groups

Conclusion:

Screening of antibiotics has been widely performed for about last 50 years and new antibiotics are still being found. In screening of new antibiotics, new approaches are required and following three factors must be considered i.e. detection of antibiotic producing microorganisms, selection of producing microorganisms and cultivation methods. These are closely related to each other, and their efficient combination is essential for successful screening of an antibiotic.

The strain A-4 and A-4 mutant was found to be having better antimicrobial activity in comparison with other soil isolates of actinomycetes, which have been investigated. The strain A-4 was mutated by UV radiation technique and drug resistance technique and the more stable strain, A-4 mutant showing maximum antibiotic production, was isolated by random screening method; and used for further experiment. The potency of the antibiotic from both A-4 and the A-4 mutant strains were compared using zone of inhibition. It is found that, A-4 mutant showed maximum potency of the antibiotic.

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Received on 15.12.2009

Accepted on 01.02.2010

Research Journal of Pharmaceutical Dosage Forms and Technology. 2(1): Jan. –Feb. 2010, 32-36