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Isolation And Diversity Of Actinomycetes

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CHAPTER -II

ISOLATION AND DIVERSITY OF ACTINOMYCETES

2.1 INTRODUCTION

Actinomycetes are heterogenous group of microorganisms holding unicellular as

well as filamentous organisms. Actinomycetes are present in huge numbers in soil.

They form the majority of microbial load of the agricultural, compost and garden soils.

They encompass highly useful enzyme and antibiotic producers to harmful disease

causing bacteria also. Actinomycetes are known to produce humic acid like

substances which improve the fertility of soil.

These are Gram positive microorganisms placed in Group 22 to 29 in Bergey’s

Manual of Determinative Bacteriology [1984]. All Actinomycetes are placed in Class

Actinobacteria of Phylum 14 in Domain II, Bacteria in Volume 4 of Bergey’s Manual of

Systematic Bacteriology [2001]. They have high GC content (around 70%).

Soil is a complex environment providing nutritional, physical and biological

variability. It serves as a complete source of nutrients for the survival of microbes.

Streptomycetes are among the most numerous and ubiquitous soil bacteria. They are

crucial in this environment because of their broad range of metabolic processes and

biotransformation. These include degradation of the insoluble remains of other

organisms, such as lignocellulose and chitin (among the world’s most abundant

biopolymers), making Streptomycetes central organisms in carbon recycling.

Actinomycetes have huge adaptability to survive in different environments. Numerous

actinomycetes were isolated from termite’s gut and their abundance was more than

bacteria [Watanabe et al., 2003]. Although they are present in large numbers in all

habitats but their number varies with season. In some parts of the world they are

more prevalent in dry season as compared to rainy season as in lakes of the Middle

Plateau, Yunnan, China. [Jiang and Xu, 1996].

Investigators all over the world have isolated Actinomycetes for various

applications and they have found immense potential and diversity in all the


47

continents. These filamentous bacteria have been able to colonize geographically

distinct locations like plain lands, agricultural soils, compost soil, river waters,

estuaries, oceans, mountains, snow covered Arctic regions etc. Their presence is

observed by classical screening methods and also based on 16S rRNA gene sequence

analysis [Lyutskanova et al., 2009; Dhanasekaran et al., 2009; Jeffrey et al.,

2008; Lo et al., 2002; Song et al., 2001].

Microbiologists believe that there is an equal amount if not more of microbial

diversity in deserts. Actinomycetes with motile spores and easy dispersal are

widespread in arid regions. Molecular ecology study indicates that only 1-5% of these

microbial species have been isolated [Hunter et al., 1998].

Marine environment has emerged as unexplored treasure for novel

Actinomycetes. The strains isolated from benthic waters are usually rare and

uncultured. The marine actinobacteria have proved to be potential sources of novel

natural products. The advent of molecular techniques and metagenomics has

accelerated the explorations. Actinobacteria emerge as an often significant, sometimes

even dominant, environmental clade in marine samples. We are yet to understand

their growth requirements. Many of the marine isolates require seawater for growth

showing high degree of marine adaptation. Exploring marine samples has led to the

isolation of more number of novel actinobacteria. Actinomycetes are also present in

many free-swimming marine vertebrates and invertebrates, as well as in sessile ones.

Cultivation of Actinomycetes sampled from a range of depths and sediments has

expanded the diversity that has been detected, extended the data available for

actinomycete classification and biogeography, and for the estimation of actinomycete

numbers [Ward and Bora, 2006].

Bredholt et al., (2008) reported the dominance of Streptomycetes and

Micromonospora in Marine water samples from Norway. Takizawa et al., (1993) also

noticed the dominance of Micromonospora in Chesapeake Bay.

The extreme variety in oceanic environments of pressure, salinity, temperature

and nutrients enable marine microorganisms to develop unique biochemical and

physiological competence for survival. This potentially offers an abundance of

secondary metabolites that might vary from the metabolites produced by terrestrial


48

microorganisms. Metagenomics enables direct access to the genome complex of

marine ecosystems for heterogeneous gene expression and functional exploitation of

uncultivable marine microorganisms [Li and Qin, 2005].

Cotarlet et al., (2010) demonstrated that Actinomycetes are not only adapted

to distinct geographical locations but also take care of their role as decomposers by

producing enzymes like protease and amylase in polar regions.

Handling, treatment and selective isolation procedures can enhance the

recovery of majority of actinomycetal types present in the given samples. The samples

used for isolation should be properly dispersed as the microorganisms showing

mycelial growth may be bound to aggregate soil particles.

Actinomycetes are slow growers as they have a typical life cycle. They produce

mycelium from spores which anchors in the substratum. The substrate hyphae are

approximately 0.5 to1.0 µm in diameter and often lack cross-walls during the

vegetative phase. Growth occurs at the hyphal apices and is accompanied by

branching, thus producing a complex tightly woven matrix of hyphae during the

vegetative growth phase. These then form aerial mycelium which bears spores. The

aerial hyphae are hydrophobic in nature. There exists a correlation between the

appearance of aerial mycelium and production of secondary metabolites such as

antibiotics.

Various kinds of sample treatments can also be done for selective isolation.

Investigators have reported selective isolation of Actinomycetes by treatment with

phenol, calcium carbonate and various antibiotics. Drying, heating and addition of

bacterial inhibitors like antibiotics are commonly used strategies for suppressing the

growth of other categories of microorganisms. Isolation of rare Actinomyces was done

by Srinivasan et al. (2001) by dry heating the sample at 120°C for 1 hr and plated on

a medium containing inorganic salts, arginine, glucose and antibiotics such as

penicillin and nystatin to isolate. Aerial spore mass is resistant to desiccation by heat

treatment. Streptomycetes are characterized by abundant sporulation but the spores

of some rare genera [Microbispora and Streptosporangium] survive heating more than

the abundantly sporulating Streptomycetes.


49

Li et al., (2002) isolated Actinomycetes from soil using extremely high

frequency radiations. This increased the frequency of isolation of rare genera by 2 to 7

times. The rare actinomycete genera were represented by Actinomadura,

Microtetraspora, Nonomuraea, Micromonospora, Amycolatopsis, Pseudonocardia,

Saccharotrix, and Streptosporangium.

Antibiotics are incorporated in the media to restrict the growth of commonly

appearing Streptomyces. Different genera are known to respond at different

concentrations of antibiotics formed the basis for the selective isolation. About 10 to

25 μg of oxytetracycline inhibited Streptomyces sp., but Streptoverticillium were

resistant to this concentration.

Most of them are aerobic and spore forming bacteria which can grow on

common laboratory media like nutrient agar with tough and leathery colonies without

aerial mycelium. They can grow on very poor media like water agar also. Their

isolation can be done by using aspargine or proline rich media. Some Actinomycetes

genera are anaerobic or require very specialized growth media and incubation

conditions eg. Frankia. They exhibit different pattern of growth on different media.

Seong et al., (2001) used hair hydrolysate vitamin agar for isolation of rare

Actinomycetes. Macromolecules such as casein, chitin, hair hydrolysate, and humic

acid can be used as carbon and nitrogen sources of rare Actinomycetes. Phenol

treatment of soil suspension also lowers the number of fungi and other bacteria, but

the Actinomycetes are less affected, thus 65% of the colonies belonged to rare

Actinomycetes.

Actinomycetes require special media to allow differentiation and development of

characteristic spores and pigments. The components present in the media or the

quality of agar also influences the good expression of taxonomic characteristics by

Streptomycetes. Highly purified agar can be devoid of growth promoters like salts of

magnesium, iron, manganese, and zinc which slow down the growth and proper

differentiation [Okami et al., 1963]. Some medium ingredients also enhance the

production of melanin pigment [Lakshmipathy et al., 2010]. Actinomycetes produce

a variety of pigments. This ranges from yellow, orange, red, purple, blue, olive green

etc. Investigators have studied them for commercial production of pigments. A well


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studied member of this group Streptomyces coelicolor produces blue pigment

[Marroquin et al., 1954].

We have attempted the isolation from a variety of samples because we wanted

to understand the diverse population of Actinomycetes in western region of Madhya

Pradesh (India) and built a broad base for screening of glucose isomerase producing

Streptomyces sp. from the nature. Our main objective is to develop glucose isomerase

production technology using indigenous isolate.

2.2 MATERIALS AND METHODS

2.2.1 Sampling

Samples were collected from various compost pits, open fields, agricultural soil

and natural vegetation soil. Altogether 26 samples (Table. 2.1) were screened for

isolation of Actinomycetes. These samples were mainly picked up from western region

of Madhya Pradesh (India) to study the actinomycetal diversity of this region. The soil

samples were picked up in commercially available sterile polythene bags from a depth

of 10 inches from surface.

The samples were named according to the sources from which they were

obtained. The isolates were named by the initial letter of the sample and numbered as

subscript. The details of samples are listed in Table 2.1

Table 2.1: List of sampling sites and number of isolates obtained

Sample Number Name Abbreviated Name No. of Isolates

Sample No. 1- Parawadi P 2

Sample No. 2- Vadelao V 8

Sample No. 3- Gamna Ga 4

Sample No. 4- Gyanpura Gy 2

Sample No. 5- Gunavad Gu 5

Sample No. 6- NRCS N 2

Sample No. 7- Biofertiliser from nearby rural area R 2


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Sample No. 8- Krishi Vigyan Kendra[Kasturba Gram] KV 1

Sample No. 9- Krishi Kshetra[Kasturba Gram Compost pit] KC 8

Sample No. 10- Krishi Kshetra[Kasturba Gram Biogas plant] KB 4

Sample No. 11- Maharaja Ranjit Singh College, Biofertiliser M 3

Sample No. 12- Monica Jain Garden Biofertiliser MJ 2

Sample No. 13- Bhaislay BI -

Sample No. 14- Bhaislay BII 1

Sample No. 15- Nandini Phanse Ma’m NPI 5

Sample No. 16- Nandini Phanse Ma’m NPII 5

Sample No. 17- Nandini Phanse Ma’m NPIII -

Sample No. 18- Kirti Singh [Farm Compost] K 1

Sample No. 19- Maharaja Ranjit Singh College, Biofertilizer MR 1

Sample No. 20- Satya Narayan [Compost pit soil] S 3

Sample No. 21- Vikas Jat [Compost pit soil] VJ 2

Sample No. 22- Abhishek Gautam [Compost pit soil] AI 2

Sample No. 23- Abhishek Gautam [Compost pit soil] AII 5

Sample No. 24- Kunjbihari Nagar [Compost pit soil] KNI 2

Sample No. 25- Kunjbihari Nagar [Compost pit soil] KNII 4

Sample No. 26- Plant soil in the lab LP 1

2.2.2 Isolation Procedure

The samples were sun dried and treated with calcium carbonate. The soil

samples were diluted in a ratio of 1:10 and 0.1 mL of the suspension was streaked on

Actinomycete isolation agar and Bennett’s agar (Appendix - I). The isolation plates

were incubated at 28°C for a week and observed daily. The Actinomycetal colonies

were purified further and observed. Their cultural characters were studied by growing


52

them on different media. Aerial spore mass colour, colony reverse (colour of substrate

mycelium), soluble pigmentation were the main features used for differentiation

among the isolates. The purified isolates were preserved on Actinomycete isolation

agar and Bennett’s agar slants.

2.2.3 Morphological analysis

The spore arrangement pattern of actinomycetes was studied by performing

slide culture technique [Williams et al., 1989]. The pure cultures were spot

inoculated on Bennett’s agar plates. A cover slip rinsed with ethanol was inserted in

the centre of the spot in inclined position. On development of the colonies, the cover

slip was removed and observed under the microscope at high power magnification

(40x).

2.2.4 Sugar utilization and enzyme characterization

The isolates were screened for the production of glucose isomerase

simultaneously and the efficient GI producers were taken up for sugar and enzyme

characterization. The cultures selected were P1, AII4, NPI2, KB1, V5.

Multiwell tissue culture plates were used for checking sugar utilization. Phenol

broth (Appendix - I) without any sugar source was prepared and 1mL of this was

poured in all the wells aseptically. Different sugar discs were added in the wells. The

cultures were inoculated and plates incubated at 28°C. The sugar utilization was

observed by the presence of growth in the respective wells.

The capacity of isolates to produce enzymes was determined by incorporating

respective substrates in Bennett’s agar medium. The isolates were spot inoculated in

the centre of the plates. Amylase production was checked by adding starch in

Bennett’s agar and cellulase by adding cellulose in Bennett’s agar [Appendix - I], the

zones were visualized by flooding the plates with iodine solution [Kar and Ray, 2008;

Kasana et al., 2008]. Gelatinase production was checked by adding gelatin in the

Bennett’s agar medium and the hydrolysis zones were visualizing by pouring

Coomassie brilliant blue on the plates [Vermelho et al., 1996]. Caseinase production

was observed by incorporating casein in the medium [Appendix - I]. Pectinase

production was checked by adding pectin in the medium and the hydrolysis zones


53

were visualized by pouring alcoholic CTAB [Appendix - II]on the plates and incubating

it for 10 minutes [Saadoun et al., 2007; Kobayashi et al. 1999].

Nitrate reduction test was performed in peptone nitrate broth (Appendix - I) to

check the ability of the isolate to reduce nitrate to nitrite and other nitrogenous

compounds. The tubes were incubated to observe the colour change by

microorganisms. Catalase test was performed by adding a few drops of hydrogen

peroxide on the organism’s growth on Bennett’s agar plate.

2.3 RESULTS AND DISSCUSSION

2.3.1 Isolation

A total of 75 cultures were isolated from 26 samples of compost pit, garden soil

and agricultural areas. The screening plates inoculated with soil samples showed 30

to 40 colonies per plate on an average. The appearance frequency of actinomycetal

colonies from the calcium carbonate treated samples was higher than those from non

treated samples. Their characterization was done on the basis of cultural characters,

biochemical characters, spore arrangement pattern and 16S rRNA sequence.

Actinomycetes grew not before 4 days of incubation at 28°C on primary screening soil

plates. Once isolated, they grew fast as pure cultures. Isolates were identified as

Actinomycetes by their chalky, velvety and powdery appearance on primary screening

plates. Their substrate mycelium could be observed developing within 48 hours. The

appearance of colonies between 2nd to 4th day seems to be like a typical bacterial

colony. This is because of the formation of substrate and aerial mycelia but no

sporulation. Confirmation of an actinomycetal colony can be done by observing the

leathery texture of the colony. The colonies are tightly held on the agar surface like a

plant on the soil surface.

Many researchers have suggested different methods for selective isolation of

Actinomycetes [Williams and Davies, 1965], where as we did not incorporate any

antibiotic in the media to restrict the growth of any kind of Actinomycetes. We wanted

to develop a collection of diverse Actinomycetes emphasizing on Streptomycetes.

Treatment of sun dried samples with calcium carbonate gave better counts as also

reported by earlier investigators [El-Nakeeb and Lechevalier, 1963]. These two

pretreatments must be decreasing the vegetative cell number and enriching the

alkalophillic spore bearing organisms.

membrane filter method for selective isolation of Actinomycetes where only the

mycelial growing forms will penetrate through the membrane filter and reach the agar

surface. They observed that the filters of pore size range of 0.45 to 0.22 allowed the

exclusive penetration of Actinomycetes only.

Fig. 2.1a is a primary screening plate which is showing numerous Actinomycete

colonies with some nonmycelial bacteria also. Streptomycetes dominated in all the

samples used for isolation which supports earlier reports of Streptomycetes being fast

growing among other Actinomycetes and predominating in the soil microbial load.

They are reported to dominate the counts in marine waters also

2007]. Fig. 2.1b is showing isolated culture picked up from a primary screening plate.

The isolates were picked u

Bennett’s agar. The samples were rich in diverse actinobacteria which is evident from

the collection of isolates.

Fig. 2.1a: Primary screening plate showing mixed colonies from soil sample, 2.1b: Isolation plate ofStreptomycete isolate P1.

Fig. 2.1a


must be decreasing the vegetative cell number and enriching the

ophillic spore bearing organisms. Hirsch and Christensen


ms will penetrate through the membrane filter and reach the agar


exclusive penetration of Actinomycetes only.

is a primary screening plate which is showing numerous Actinomycete



r Actinomycetes and predominating in the soil microbial load.

They are reported to dominate the counts in marine waters also

Fig. 2.1b is showing isolated culture picked up from a primary screening plate.

The isolates were picked up and transferred repeatedly to get pure cultures on

The samples were rich in diverse actinobacteria which is evident from

the collection of isolates. Some of the representatives are shown in

Fig. 2.1a: Primary screening plate showing mixed colonies from soil sample, 2.1b: Isolation plate ofP1.


54

must be decreasing the vegetative cell number and enriching the

Christensen, (1983) proposed


ms will penetrate through the membrane filter and reach the agar


is a primary screening plate which is showing numerous Actinomycete



r Actinomycetes and predominating in the soil microbial load.

They are reported to dominate the counts in marine waters also [Ramasamy et al.,

Fig. 2.1b is showing isolated culture picked up from a primary screening plate.

p and transferred repeatedly to get pure cultures on

The samples were rich in diverse actinobacteria which is evident from

shown in Fig 2.2.

Fig. 2.1a: Primary screening plate showing mixed colonies from soil sample, 2.1b: Isolation plate of

Fig. 2.1b


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Fig

.2.2

:A

few

repre

sen

tati

ves

of

the

Acti

nom

yceta

lis

ola

tes


56

2.3.1.1 Aerial spore mass appearance

The isolates produced a range of aerial spore mass colours, colony reverse and

pigments. The aerial mycelium and spore mass were white (N1, N2, VJ2), creamish or

ivory (V6, Ab, Gu3, KC4), yellowish cream (Gu1), orangish (V2, V3, NPII4), pink (Gu2,

P) glossy green (NPI6), purplish grey (NPII1), dark brown (V1, V4), orangish brown

(MR1), light grey (P1, P2, V5, MJ1) and dark grey (KB4, AII4), blue (NPI5, NPII5.) and

black (S4) in colour. The colonies appeared leathery till the development of substrate

mycelia but gained powdery, cottony, velvety or ash like appearance as aerial spore

mass developed. A photographic representation of these categories have been shown

in Fig. 2.3a-i and Fig. 2.4 a-i

Fig. 2.3a

Fig. 2.3e

Fig. 2.3b

Fig. 2.3d

Fig. 2.3c

Fig. 2.3f


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Fig. 2.3 Varied spore mass colour a: VJ2, white; b: Gu3, ivory; c: S4, black; d: LP, pink; e: pink; f:V4, dark brown; g: P1, ash grey; h: P2 light grey; i: NPII1, bluish grey.

Fig. 2.4 a: Dark velvety grey spore mass colour of AII4; b: Very dark grey spore mass colour of KB4;c: light blue spore mass colour of NPI5; d:blue spore mass colour of NPII5; e:glossy green coloniesof NPI6.

Fig. 2.3g Fig. 2.3iFig. 2.3h

Fig. 2.4a Fig. 2.4b

Fig. 2.4c Fig. 2.4d Fig. 2.4e

2.3.1.2 Pigmentation

The collection of isolates was diverse with respect to growth pattern, aerial and

substrate hyphae and pigments.

observed among the isolates.

the isolates. Our collection had some isolates which exhibited light coloured aerial

spore mass and produced abundant soluble pigment contrasting the jet black colour

spore mass possessing isolate which did not produce any pigment.

and V4 showed heavy olive green pigmentation in the beginning which later turned

colour of the whole med

production is influenced by medium composition and pH

Zapata, 1954]. The pigmentation was favored on Actinomycete isolation agar and

Bennett’s agar. The isolates

KC3, KC4 pale yellow, KC1 light pink, M2, M3 light purple, KC5 brown, KNI1 dark

brown KNII3 dark purlish black, NPII1 mustard

produced the earthy odor of Geosmins

1965].

Fig. 2.5:


ation


hyphae and pigments. Excessive to moderate pigment production was also

the isolates. Production of melanoid pigments was widespread among


roduced abundant soluble pigment contrasting the jet black colour

spore mass possessing isolate which did not produce any pigment.

and V4 showed heavy olive green pigmentation in the beginning which later turned

colour of the whole media to dark brown. As reported by earlier researchers

production is influenced by medium composition and pH [

. The pigmentation was favored on Actinomycete isolation agar and

The isolates V2 and V3 produced purplish pink pigment, NPI2, V6, Ab,


purlish black, NPII1 mustard [Fig. 2.5].

produced the earthy odor of Geosmins in pure cultures [

: Different coloured pigments produced by isolates.


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igment production was also

Production of melanoid pigments was widespread among


roduced abundant soluble pigment contrasting the jet black colour

spore mass possessing isolate which did not produce any pigment. The isolates V1

and V4 showed heavy olive green pigmentation in the beginning which later turned the

ia to dark brown. As reported by earlier researchers, pigment

[Sanchez-Marroquin and

. The pigmentation was favored on Actinomycete isolation agar and

V2 and V3 produced purplish pink pigment, NPI2, V6, Ab,


Majority of the cultures

[Gerber and Lechevalier,

ifferent coloured pigments produced by isolates.


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2.3.1.3 Colony reverse

Many cultures looked same from the colony surface appearance but they were

different when observed from the reverse of the colony. The colony reverse showed the

difference in the substrate hyphae. The cultures developed as rubbery, hard or chalky

white colonies in the beginning which later on developed into their original forms

showing velvety or powdery appearance with respective colours of aerial mass. The

colony reverse of isolates were yellow (Ga), mustard (V6), orange (V3), reddish brown

(P1, P2), grey (KB1) and white (Gu). This formed the basis of primary differentiation of

cultures. A vast range of colony reverse among the isolates can be observed in Fig. 2.6.

Fig. 2.6: Actinomycetal isolates showing different mycelial colour (colony reverse).

The colonies developed concentric rings on aging [Fig. 2.7]. There was a slight

difference in colour in the centre and in the concentric rings. The growing ends were

lighter in colour. Most of the Streptomyces isolates exhibited white colonies which

became grey on aging.

Fig. 2.7: Appearance of concentric rings around the

2.3.1.4 Colony surface appearance

There was a huge variety in the texture of

had grey aerial spore mass, some had powdery ash like texture (P1), some dark velvety

grey (AII4), some formed webbed colonies

appearance. The isolated colonies of VJ1

surface with depression in the centre of the colony.

drops on the surface of the colonies

Fig. 2.7a

Fig. 2.7d


2.7: Appearance of concentric rings around the isolates on aging.

Colony surface appearance

There was a huge variety in the texture of Streptomyces


grey (AII4), some formed webbed colonies [Fig. 2.8a] and others showed cottony

appearance. The isolated colonies of VJ1 [Fig. 2.8b] exhibited

surface with depression in the centre of the colony. The isolate

drops on the surface of the colonies [Fig. 2.8c].

Fig. 2.7d

Fig. 2.7b


60

isolates on aging.

Streptomyces isolates which definitely


and others showed cottony

exhibited wavy appearance on the

The isolate KB4 produced dew

Fig. 2.7e

Fig. 2.7c


61

Fig. 2.8a: Webbed colonial appearance; b: depressions on the top of the colonies; c: dew drops

produced on the colony surface.

2.3.1.5 Growth response on different media

The isolates were grown on a different media (Actinomycete isolation agar,

Bennett’s agar and Wheat bran agar) to study their growth pattern. The media

composition was found to influence the extent of sporulation and appearance of aerial

mycelia which is in accordance with earlier researchers [Okami et al., 1963;

Sanchez-Marroquin, 1962]. They also differed in the time required for sporulation

and attaining the spore colour on maturity. Bennett’s agar medium was helpful in

understanding the cultural characters like aerial spore mass colour, colony reverse

and pigmentation satisfactorily while wheat bran agar was useful for fast growth of the

organism as it is an enriched medium. Sporulation occurred very fast with dense

growth on this medium which is in accordance with earlier reporters [Manhas and

Bala, 2004; Srinivasan et al., 1983]. Fast and luxurious growth of the cultures on

wheat bran medium must be due to the elaborate nutrients provided by wheat bran.

Graying of the Streptomycetes colonies was also accelerated on this medium. A

comparison of growth of isolates on different media is shown in Fig. 2.9. The detailed

cultural characters of 75 actinomycetal isolates are mentioned in the Table 2.2.

Fig. 2.8a Fig. 2.8b Fig. 2.8c


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Fig 2.9 Different appearance of same isolate on different media; a(i): NPII2 grown on Actinomycete

isolation agar; a(ii): NPII2 grown on Wheat bran agar, a(iii) NPII2 grown on Bennett’s agar; b(i):

NPI2 grown on Actinomycete isolation agar; b(ii): NPI2 grown on Wheat bran agar; b(iii) NPII2

grown on Bennett’s agar.

Table 2.2: Cultural characters of actinomycetal isolates

Isolate Aerial Spore

Mass

Texture Colony Reverse Soluble Pigment

P1 Ash Grey Powdery Blakish brown Light grey

P2 Dark Grey Powdery Brownish grey -

V1 Dark Ash Grey Ashlike Black Dark Olive Green

V2 Purplish Grey Velvetty Purplish Black Light purple

V3 Purplish Grey Velvetty Purplish Black Light purple

V4 Dark Ash Grey Ashlike Black Dark Olive Green

V5 Light Grey Cottany Mustard brown Mustard grey

Fig. 2.9a Fig. 2.9b

iii i ii

iiiiii


63


Mass


V6 Dirty White Chalky Mustard Pale

Ab Dirty White Chalky Mustard Pale

KB1 Light Grey Ashlike Brown Grey

KB2 Whitish Grey Powdery Dark Brown Light Grey

KB3 Light Ash Grey Chalky Creamish -

KB4 Greenish Grey Ashlike Greenish Mustard Mustard black

M2 Dark Grey Powdery Dark Brown Greyish Purple

M3 Light Grey Powdery Brick Brownish Blakish Grey

M4 Light Grey Powdery Blakish Brown Blakish Grey

Ga1 Dirty Yellowish

White

Powdery Yellow -

Ga2 White Fibrous White -

Ga3 White Chalky White -

Ga4 White Chalky Brownish Purple -

Gy1 Light Grey Powdery Blackish Brown -

Gy2 Light Grey Powdery Mustard Light Orange

Gu1 Creamish

Yellow

Chalk Powder White -

Gu2 Purplish pink Powdery Peach Violet

Gu3 Yellowish Grey Velvety Greenish yellow -


64


Mass


Gu4 Light Grey Velvety Yellow -

Gu5 Light Grey Powdery White Creamish

R1 Grey Powdery Blakish Grey Light Mustard

R2 White Fibrous White -

N1 White Chalky Mustard Mustard

N2 White Chalky White -

KC1 Creamish

Yellow

Fibrous Pinkish Brownish

KC2 Grey Velvetty Blackish Brown Purpulish Grey

KC3 Yellowish Grey Powdery Light Mustard -

KC4 Dirty Yellowish

Grey

Powdery Light Mustard Light Mustard

KC5 Bluish Grey Velvetty Blakish Grey Light Grey

KC6 Grey Velvetty Black Greyish Purple

KC7 Light Grey Cottany Pale -

KC8 Yellowish White Powdery Light Mustard Pale

MR1 Grey Velvetty Mustard Grey -

NPI1 Dirty White Cottany Blakish Brown Greyish Purple

NPI2 Grey Velvetty Purpulish Mustard Light Purple

NPI3 Dirty White Cottany Blakish Brown Light Purple


65


Mass


NPI4 White Chalky White -

NPI5 White Chalky White -

NPI6 Bluish Grey Velvetty Grey -

NPII1 Earthy Brown Powdery Blakish Brown Purplish Black

NPII2 Grey Powdery Blakish Brown Light Purple

NPII4 White Cottany Light Mustard -

NPII5 Bluish Grey Velvetty Bluish Grey -

NPII6 Green Glossy Grey -

KNI1 Ash Grey Ashlike Black Greyish Brown

KNI2 Light Grey Powdery Grey -

KNII1 Earthy Brown Powdery Blakish Brown Purplish Black

KNII2 Buff Grey Velvetty Light Mustard Light Purple

KNII3 Dark Greyish

Purple

Powdery Dark Brown Brownish Purple

KNII4 Buffy White Chalky White -

AI1 Dark Ash Grey Powdery Grayish Black -

AI2 Dark Grey Powdery Brown Brownish

AII1 Grey Powdery Blakish Brown -

AII2 White Chalky White -

AII3 White Fiborous White -


66


Mass


AII4 Ash Grey Velvety Mustard with Grey

Margin

-

AII5 Blue Chalky Grey Light Purple

BII1 Dirty White Chalky Dark Grey -

MJ1 Grey Powdery Greyish Mustard -

MJ2 Grey Chalky Greyish Mustard -

VJ1 Light Grey Velvety Orangish Mustard -

VJ2 White Chalky White -

S1 White Chalky White -

S2 Grey Velvety Dark Grey -

S4 Black Velvety Black -

KV White Chalky White -

LP Pink Velvety Pink -

K White with grey

spots

Powdery Greyish -

2.3.1.6 Growth in liquid media

The actinomycetes grew in liquid media (Bennett’s broth and Medium No. 9) in

the form of beads. The size and appearance of beads varied with the media

composition and type of the organism. Fig. 2.10 shows the growth of the isolate P1 in

different media combinations. The beads appear as orange and light brown in Medium

No. 9 (Fig. 2.10a) whereas it appears white in Bennett’s broth as in Fig. 2.10b. The

fibrous outgrowth from the beads to varying degrees is visible in Fig. 2.10c, d and e.

The beads in Fig. 2.10f are absolutely smooth.


67

Fig. 2.10: Actinomycetes growing in liquid media.

Fig. 2.10a

Fig. 2.10c Fig. 2.10d

Fig. 2.10b

Fig. 2.10e Fig. 2.10f

2.3.2 Morphological Analysis

The mycelium and spore pattern

technique [Williams et al., 1989]

during the initial phase of sporulation.

mycelium can be differentiated from darker less branched aerial mycelia.

mycelia are hydrophobic in nature and stan

between the appearance of aerial mycelium and production of secondary metabolites

such as antibiotics. Most of the antibiotic producing strains are known to be

pigmented.

The pattern of spore arrangement was studied a

according to the features described in Bergey’s Manual of Systemic Bacteriology

Volume 4 [2001].

The isolates P1, P2, V1, V4, KB1, KB4, M2, Gy1, KC2, KNI1, MJ2

exhibited typical spiral spore arrangement as of

Ab and KC3 were grouped as

The isolates Ga3, Gu1, KC1 showed straight chains of spores with curled ends as

Actinomadura. Pseudonocardia

Saccharomonospora had a series of doubly placed spores as in the isolates NPI5, NPI6

and AII5 (Fig. 2.11g

branched ends and were placed in

Micromonospora had one spore at the terminus as in

Fig. 2.11a


Morphological Analysis

The mycelium and spore pattern can be easily studied by slide culture

[Williams et al., 1989]. The isolate morphology can be easily understood

during the initial phase of sporulation. At this stage the densely

be differentiated from darker less branched aerial mycelia.

hydrophobic in nature and stand upright. There exists a correlation



The pattern of spore arrangement was studied and the isolates were grouped


The isolates P1, P2, V1, V4, KB1, KB4, M2, Gy1, KC2, KNI1, MJ2

typical spiral spore arrangement as of Streptomyces

Ab and KC3 were grouped as Kitasatosporia as they showed straight chains of spores.

Ga3, Gu1, KC1 showed straight chains of spores with curled ends as

Pseudonocardia exhibited wavy chains of spores

had a series of doubly placed spores as in the isolates NPI5, NPI6

2.11g). The isolate AII3 had straight spore chains developing from

branched ends and were placed in Streptoverticillium group.

had one spore at the terminus as in the isolate

Fig. 2.11bFig. 2.11a


68

be easily studied by slide culture

isolate morphology can be easily understood

densely branched substrate

be differentiated from darker less branched aerial mycelia. The aerial

There exists a correlation



nd the isolates were grouped


The isolates P1, P2, V1, V4, KB1, KB4, M2, Gy1, KC2, KNI1, MJ2 and AII4

yces (Fig. 2.11a). The isolates

as they showed straight chains of spores.

Ga3, Gu1, KC1 showed straight chains of spores with curled ends as

exhibited wavy chains of spores as in Ga4 and BII1.

had a series of doubly placed spores as in the isolates NPI5, NPI6

AII3 had straight spore chains developing from

group. The mycelium of

the isolate MR1.

Fig. 2.11b

Fig. 2.11c

Fig. 2.11e


Fig. 2.11c Fig. 2.11d

Fig. 2.11e Fig. 2.11f


69

Fig. 2.11d

Fig. 2.11f

Fig. 2.11i

Fig. 2.11g


Fig. 2.11i

Fig. 2.11g

Fig. 2.11j


70

Fig. 2.11h

Fig. 2.11j

Fig. 2.11: Microscopic details of spore arrangement pattern of a few isolates

(a-f): a: P1; b: AII4; c:

Streptoverticillium

Streptomyces -

Dermatophillus -

Fig. 2.11k

Fig. 2.11m


Fig. 2.11: Microscopic details of spore arrangement pattern of a few isolates

P1; b: AII4; c: V5; d: KB1; e: KB2; f: Gy2; g: Saccharomonospora

Streptoverticillium - AII3; i: Nocardioides - VJ1; j: Streptoverticillium

KC5; l: Thermomonospora - P; m: Saccharomonospora

- S1.

Fig. 2.11k Fig. 2.11l

Fig. 2.11mFig. 2.11n


71

Fig. 2.11: Microscopic details of spore arrangement pattern of a few isolates, Streptomyces

Saccharomonospora - AII5; h:

Streptoverticillium- KC8; k:

Saccharomonospora - NPI6; n:

Fig. 2.11l

Fig. 2.11n


72

2.3.4 Biochemical characterization

Sugar utilization was checked for five good producers of glucose isomerase.

Glucose, raffinose and terhalose, xylose and sucrose were found to be the sugars of

choice of the isolates [Table 2.3] [Pridham and Gottlieb, 1948]. According to the

description given in Bergey’s Manual of Systemic Bacteriology [2001], most of the

Streptomyces of gray aerial spore mass series utilize glucose and xylose whereas

raffinose and sucrose is not very commonly utilized. The isolates P1, AII4, V5 and KB1

were positive for catalase whereas NPI2 was found to be negative. All the isolates

exhibited cellulolytic, lipolytic and pectinolytic activity. The isolates P1, V5, KB1 and

NPI2 also degraded starch but AII4 did not show amylase production [Table 2.4].

Table 2.3: Sugar Utilization tests for selected Streptomyces isolates

Sugar P1 AII4 VPI2 KB1 V5

Maltose - - + - -

Mannitol - - - - -

Mannose - + + + -

Melibiose - - - - -

Raffinose + + + + +

Rhamnose - - - - -

Salicin - - + - -

Sorbitol - - - - -

Sucrose - - - + -

Trehalose + + + + +

Xylose + + + + +

Adonitol - - - - -

Arabinose - + - + +

Cellobiose - - - + +

Dextrose + + + + +

Dulcitol - - - - -


73

Fructose - + - - -

Galactose - - + + +

Inositol - - - + -

Inulin - - - + -

Lactose - - - - -

[(+) = Sugar Utilized (-) = Sugar not Utilized]

Table 2.4: Enzymatic characterization for selected Streptomyces isolates

Test P1 A II4 NPI2 KB1 V5

Glucose isomerase production + + + + +

Amylase production + - + + +

Protease production - - - - -

Lipase production + + + + +

Cellulase production + + + + +

Gelatinase production + - - + +

Pectinase production + + + + +

Nitrate reductase + - + + +

Catalase + + - + +

(+) = Enzyme produced (-) = Enzyme not produced

2.4 CONCLUSIONS

The isolation of morphologically and culturally diverse actinobacteria has

revealed a treasure of bioresource in the western region of Madhya Pradesh. The

presence of huge counts of actinomycetes in soil also indicates the fertility of soil, as

these organisms are known to be efficient in mineralization and recycling of the

organic matter. The samples yielded different group of organisms but the grey spore

mass bearing Streptomycetes was dominant. The growth rate of grey series was quiet

high as compared to white and blue spore mass bearing isolates. The sporulation was

also better by Streptomycetes as compared to non-Streptomycete isolates.


74

The objective behind screening diverse actinomycetes was to point out an

efficient glucose isomerase producing Streptomycete strain. Streptomycetes are widely

used for the production of glucose isomerase at industrial level. The biochemical

characters exhibited by the selected isolates and the spore pattern arrangement

studied by slide culture technique help us to conclude that these organisms belong to

the Streptomycete group. The elaborate enzyme production capacity of these isolates

accounts for the presence of all the macromolecules which are being degraded by

them. All the selected isolates had typical spiral spore arrangement which is a

characteristic feature of this group. Slide culture technique is a quick tool for

determination of the actinobacterial group. Their confirmation will be done further by

sequencing of 16s rRNA.

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