09 chapter

45

CHAPTER 4

RESULTS

4.1 ISOLATION AND IDENTIFICATION OF ACTINOMYCETES

FROM SOIL SAMPLES OF EASTERN GHATS

4.1.1 Soil Nutrient Analysis of Yercaud Hills

The results on soil nutrient analysis of Yercaud hills belonging to

eastern Ghats showed that soils were found to be neutral with a pH range of

5.9–6.1 (Table 4.1). Further, electrical conductivity (EC) was ranged between

0.17 and 0.24 dSm-1

and the total organic carbon content was varied in varying

levels of 3.84–4.90%. Soil samples obtained from the top station areas of

Yercaud hills registered with high organic carbon (4.90 %) content when

compared to soils of other sites analyzed. Available potassium content in soil

samples was determined between 228 and 292 mg/g of soil dry weight. A least

potassium nutrient level was observed in a low station (228 mg/kg) soil

samples. Similarly, available nitrogen and phosphorus contents were

determined between 2.11 - 2.47% and 17.86 - 21.62 mg/kg soil dry weight;

respectively. Water holding capacity (WHC) in soil samples was estimated

between 65.2 and 78.5%. The concentration of calcium was found to be higher

in top station (267 mg/kg) soils than in soil samples collected from other sites

of Yercaud hills. Available magnesium and sodium contents of soil samples

were observed between 92-108 and 20.8–26.7 mg/kg soil dry weight;

respectively.

46

4.1.2 Soil Nutrient Analysis of Kolli Hills

The pH of soil samples of Kolli hills to the same eastern Ghats

showed that it was found to be the same with that of the Yercaud soil with a pH

range of 5.6 – 6.2 (Table 4.2). Further, the EC and total organic carbon content

were ranged between 0.17 – 0.23 dSm-1

and 4.65%-4.88%; respectively in soil

samples. Available potassium content in a topographical area was recorded at

the maximum (296 mg/g) when compared to that of forest (282 mg/g of soil

dry weight) areas. A least nitrogen nutrient level was observed in a low station

(2.11 mg/kg) soil samples. Available phosphorus content determined was in the

range of 19.24 – 21.25 mg/kg soil dry weight. Water holding capacity (WHC)

in soil samples was estimated between 69.1 and 85.2%. Exchangeable calcium

concentration was found to be higher in the soils of forest areas (267 mg/kg)

than other sites of Kolli hills. Similarly, available magnesium and sodium

contents of soil samples were observed between 92 - 110 and 20.1– 25.7 mg/kg

soil dry weight; respectively.

4.1.3 Population Density of Actinomycetes in Yercaud and Kolli hills

Actinomycetes population was enumerated from soil samples

collected from various sites of Yercaud and Kolli Hills of Eastern Ghats using

a starch casein nitrate (SCN) agar medium by following serial dilution

technique. Population density of actinomycetes in rhizosphere and non-

rhizosphere soils of various medicinal plants revealed that it was found to be

higher in the rhizosphere than in non-rhizosphere regions. Moreover, it was

found to be more in top station when compared to other sites of Yercaud and

Kolli hills (Tables 4.3 and 4.4). The population was found to be 34.1×102 /gm

soil dry weight in top station site, followed by topographical area (30.8 gm soil

dry weight). A least population load was recorded in the terrain region of both

rhizosphere and non-rhizosphere regions (21.9 and 13.4×102 /gm soil dry

weight; respectively). With respect to Kolli Hills, the least population density

was recorded with forest areas which accounted as 12.3×102 /gm soil dry

weight.

47

Table 4.1 Physico-chemical properties and population density of Actinomycetes in soils of Yercaud hills of Eastern

Ghats

Sampling

SitespH EC OC WHC N P K Na Ca Mg

Population of

Actinomycetes#

Top station 5.5 0.22 4.90 74.6 2.78 21.62 296 30.4 267 120 32.1

Forest areas 6.1 0.19 4.31 71.6 2.37 20.27 267 24.3 250 108 22.5

Terrain 6.0 0.21 3.84 68.8 2.11 19.84 260 25.5 252 102 09.4

Low station 5.9 0.24 4.38 67.8 2.45 20.86 258 26.7 256 97 18.6

Topographical

area6.0 0.17 4.62

66.92.27 19.32 282 23.5 245 99 22.4

SE±

CD at P =0. 050.17 0.04 0.12 1.14 0.07 0.72 4.81 0.81 7.28 5.64 1.03

0.30 0.09 0.27 2.44 0.15 1.62 9.74 1.48 16.22 11.90 0.28

EC - Electrical conductivity (dSm-1

) OC - Total organic carbon (%)

WHC - Water holding capacity (%) N - Total nitrogen content (%)

P - Available phosphorus (mg/kg) K - Exchangeable potassium (mg/kg)

Na - Exchangeable sodium (mg/kg) Ca - Exchangeable calcium (mg/kg)

Mg - Exchangeable magnesium (mg/kg) #

cfu × 102/gm of soil dry weight

All the data represented in the table are the mean of three replicates.

Values followed by the same alphabets are not significantly different at P< 0.05.

48

Table 4.2 Physico-chemical properties and population density of Actinomycetes in soils of Kolli Hills of Eastern Ghats

Sampling

SitespH EC OC WHC N P K Na Ca Mg

Population of

Actinomycetes#

Top station 5.0 0.20 6.5 78.9 3.2 25.6 290 29.8 280 108 30.8

Forest areas 6.1 0.12 3.27 69.1 2.27 19.68 270 24.6 260 79 25.6

Terrain 5.6 0.17 4.11 76.3 2.34 19.24 267 25.7 253 75 12.8

Low station 5.7 0.17 3.65 70.8 2.18 20.37 267 20.1 230 78 20.7

Topographical

area6.0 0.22 5.0

74.32.29 20.98 276 22.9 210 80 26.9

SE±

CD at P =0.050.15 0.06 0.11 1.12 0.04 0.62 3.69 0.79 6.28 4.96 1.18

0.29 0.08 0.29 2.55 0.09 1.12 7.15 1.48 13.67 8.56 2.65

Ec - Electrical conductivity (dSm-1

) OC - Total organic carbon (%)

WHC - Water holding capacity (%) N - Total nitrogen content (%)

P - Available phosphorus (mg/kg) K - Exchangeable potassium (mg/kg)

Na - Exchangeable sodium (mg/kg) Ca - Exchangeable calcium (mg/kg)

Mg - Exchangeable magnesium (mg/kg) #

cfu × 102/gm of soil dry weight


Values followed by the same alphabets are not significantly different at P< 0.05.

49

Table 4.3 Population density of Actinomycetes in the rhizosphere and

non-rhizosphere regions of various medicinal plants from

Yercaud hills

Sampling

sites

Population density of Actinomycetes#(×10

2/gm of soil dry wt.)

Rhizosphere

region

Non-rhizosphere

region

Top station 34.1 27.2

Forest areas 21.9 13.4

Terrain 26.0 18.3

Low station 29.2 15.8

Topographical area 30.8 21.6

SE± 3.51 4.12

CD at P =0.05 7.58 9.17 #

Mean of three replicates

Table 4.4 Population density of Actinomycetes in the rhizosphere and

non-rhizosphere regions of various medicinal plants from Kolli

hills

Sampling

sites

Population density of

Actinomycetes#(×10

2/gm of soil dry wt.)

Rhizosphere

region

Non-rhizosphere

region

Top station 31.3 22.8

Forest areas 17.2 12.3

Terrain 20.6 18.3

Low station 19.2 16.7

Topographical area 28.8 18.1

SE± 3.8 2.8

CD at P =0.05 7.2 4.2 #

Mean of three replicates

50

4.1.4 Population density of Actinomycetes in soilsamples collected

from different medicinal plants of Yercaud and Kolli hills

The population of actinomycetes was enumerated from rhizosphere

as well as from non-rhizosphere regions of important medicinal plants of

Yercaud and Kolli hills (Table 4.5 and Table 4.6). The results revealed that

the population density of actinomycetes was found to be higher in soil

samples collected from rhizosphere than in non-rhizosphere regions. It was

higher in the rhizosphere soils of Asparagus racemosus (36.2 gm soil dry

weight) followed by Andrographis paniculata (30.1 gm soil dry weight)

plants at Yercaud hills. A least population density was recorded with

Cissusquadrangularisand Gymnemasylvestre (20.3 gm soil dry weight)

plants.In Kolli hills, the population density was high in the rhizosphere region

of Acacia nilotica (30.3 gm soil dry weight) followed by Andrographis

paniculata (28.6 gm soil dry weight) plants. The population load was found to

be least with Cissus quadrangularis (18.6 gm soil dry weight) plant.

4.1.5 Distribution Pattern of Population Density of Actinomycetes in

Eastern Ghats (Yercaud and Kolli hills)

The distribution pattern of actinomycetes population in the

rhizosphere soils of different medicinal plants is presented in the Figures 4.1-

4.4. The results revealed that the population density was decreased when the

distance as well as depth of soil sampling of the plants was increased. At

175cm depth and 200 cm distance from the plants, the population of

actinomycetes was not recovered completely in both Yercaud and Kolli hills.

This is because of the close relationship between the rhizosphere region of

plants and microorganisms. The population load of actinomycetes at 25 cm

distance was found to be more (16.2×102gm/soil dry wt.) and thereafter it was

51

declined sharply when distance / depth from the plants was increased to

collect rhizosphere soil samples. Moreover, the population density was

recorded at the maximum with Asparagus racemosus and Acacia nilotica

plants in Yercaud and Kolli hills; respectively.

Table 4.5 Population density of Actinomycetes in soil samples

collected from different medicinal plants of Yercaud hills of

Eastern Ghats

Medicinal plants

Population density of Actinomycetes#

(×102/gm of soil dry wt.)

Rhizosphere

region

Non-rhizosphere

region

Asparagus racemosus 36.2 24.3

Andrographis paniculata 30.1 20.7

Solanum nigrum 29.8 21.8

Zingiber officinale 20.3 13.2

Cissus quadrangularis 28.6 18.3

SE± 3.1 2.4

CD at P =0. 05 6.7 5.2

# Mean of three replicates

52

Table 4.6 Population density of Actinomycetes in soil samples

collected from different medicinal plants of Kolli Hills of

Eastern Ghats

Medicinal plants

Population density of Actinomycetes#

(×102/gm of soil dry wt.)

Rhizosphere

region

Non Rhizosphere

region

Acacia nilotica 30.3 21.3

Adhatoda zeylancia 27.2 22.1

Andrographis paniculata 28.6 20.6

Cissus quadrangularis 28.1 18.6

Eclipta prostrate 21.8 20.4

SE± 1.8 1.1

CD at P =0. 05 3.2 2.4

# Mean of three replicates

53

Figure 4.1 Distribution pattern of Actinomycetes population with

reference to depth of different medicinal plants from

Yercaud hills

54


reference to distance of different medicinal plants from

Yercaud hills

All the data represented in the figure are the mean of three replicates

55


reference to depth from different medicinal plants from

Kolli hills

56


reference to distance from different medicinal plants from

Kolli hills

All the data represented in the figure are the mean of three replicates

57

4.2 CHARACTERIZATION AND IDENTIFICATION OF

ACTINOMYCETES FROM EASTERN GHATS

4.2.1 Characterization and identification of actinomycetes

A total of 168 strains were isolated from soil samples collected

from eastern Ghats and screened them subsequently based on their potential

of secondary metabolite production in which two isolates from each hill

(Yercaud and Kolli hills) were chosen for further studies. All the strains were

identified by following the method of Bergey’s manual, wherein, the

polyphasic taxonomic approach was adopted. All the actinomycete strains

were identified as Streptomyces sannanensis. Further identification was

confirmed by Microbial Type Culture collection Centre (MTCC), Chandigarh,

India. The results of various morphological, biochemical and physiological

tests were summarized in the Table 4.8 and 4.9. The strains such as Yer11 and

Yer28 obtained from Yercaud hills and Kol35 and Kol44 strains obtained

from Kolli hills were selected. These proven strains were designated based on

the hills and depository strain number (KSR College Microbial Collection

Depository). In addition, a standard strain (MTCC 6285) was procured and

used for comparison purpose.

The actinomycete strains were found to be spiral spore

morphology, Gram-positive bacterium and from greyish to pinkish in colour.

They were produced different colour of pigments and catalytic and oxidative

enzymes such as catalase, lipase and urease. All the strains were hydrolyzed

gelatine, casein and starch in the basal medium as well. They were negative to

indole production, methyl red and voges proskauer test but utilized citrate.

Further, the cultures were negative for oxidase production. Nitrate was

reduced very significantly and mannitol was utilized by the strains and they

utilized fructose, glucose and dextrose as well. They showed a prominent

growth at a pH of 6, 30º C temperature, 80% relative humidity and 2% NaCl

concentration. All the proven strains were grown well in 12D:12L photo

58

period. In the case of Kol35 strain, it was white in colour and spores were

found to be in cluster sometimes coiled chain form. The colony colour was

found to be creamy yellow in nature (Figure 4.5 and 4.6).

A – Yer11, B – Yer28, C – Kol35, D – Kol44, E – MTCC 6285

Figure 4.5 Colony morphology of selected actinomycete strains isolated

from Yercaud and Kolli Hills of Eastern Ghats

A

C D

E

59

A – Yer11, B – Yer28, C – Kol35, D – Kol44

Figure 4.6 Microscopic observation of selected isolates of actinomycetes

after Gram’s staining from Yercaud and Kolli hills of

Eastern Ghats

A B

C D

60

Table 4.7 Morphological, biochemical and physiological characterizations of Streptomyces spp.

Parameters Isolates of Streptomyces spp.

Yer11 Yer28 Kol35 Kol44 MTCC6285

Cell morphologySpiral spore chain Rods and coiled Cluster of spore chain

Coiled spore

ChainRods and coiled

Colour of the mycelium/colony Grayish white Pinkish white White powdery Creamy yellow White powdery

Gram’s staining + + + + +

Pigment production +++ +++ +++ +++ -

Starch hydrolysis +++ ++ - ++ ++

Casein hydrolysis +++ ++ - ++ ++

Catalase test +++ +++ +++ ++ ++

Nitrate reduction +++ +++ +++ ++ -

Indole production - - - - -

Gelatin hydrolysis ++ ++ +++ ++ +++

Hydrogen sulphide production ++ ++ +++ ++ -

Methyl red test - - - - -

Voges Proskauer test - - - - -

Citrate utilization test +++ ++ ++ ++ ++

Urease test +++ ++ ++ ++ ++

Oxidase test - - - - -

Lipid hydrolysis ++ ++ ++ ++ ++

Mannitol utilization ++ ++ ++ ++ ++

+ ++ Prominent growth, ++ Moderate growth, + Minimum growth, - No growth

61

Table 4.8 Characterization traits of Streptomyces spp.

Parameters

Isolates of Streptomyces spp.


Carbon sources

Fructose + ++ + ++ ++

Glucose ++ ++ ++ ++ ++

Dextrose ++ ++ ++ ++ +++

Nitrogen sources

Ammonium nitrate + ++ + ++ ++

Sodium nitrate ++ ++ ++ ++ ++

Potassium nitrate ++ ++ ++ ++ +++

Yeast extract + ++ + ++ ++

pH Concentration

pH 5 ++ + ++ ++ ++

pH 6 ++ ++ ++ ++ ++

pH 7 +++ +++ +++ +++ ++

pH 8 +++ +++ +++ +++ +++

pH 9 +++ +++ +++ +++ +++

62

Table 4.8 (Continued)

Parameters



Temperature (º C)

10 º C + + - - +

20 º C ++ ++ ++ ++ ++

30 º C +++ +++ +++ +++ +++

40 º C +++ +++ +++ +++ +++

50 º C ++ ++ + ++ +

NaCl Concentration

0% - - + + +

1% +++ +++ +++ +++ +++

2% +++ +++ +++ +++ +++

3% ++ ++ ++ ++ ++

4% + + + + +

63

Table 4.8 (Continued)

Parameters



Relative humidity (%)

60 % + + + + +

70 % ++ ++ ++ ++ ++

80 % +++ +++ +++ +++ +++

90 % ++ ++ ++ ++ ++

Photo periods

14 D:10 L + + + + +

12 D:12 L +++ +++ +++ +++ +++

10D:14 L ++ ++ ++ ++ ++

8 D:16 L + + + + +

+ ++ Prominent growth

++ Moderate growth

+ Minimum growth

- No growth

64

4.2.2 Standardization of Different Media for Mass Multiplication of

Streptomyces Strains

Various types of synthetic, semi-synthetic and natural media were

selected to find out a suitable medium for the maximal growth and mass

multiplication of S. sannanensis strains under in vitro condition. The

population load of S. sannanensis strains in the media was determined and

presented in the Table 4.10. The results indicated that among the various

media used, SCN medium was found to be an appropriate one for the growth

of S. sannanensis strains. The maximum growth was obtained in the strain of

Yer28 (12.5 × 102/mL) and followed by MTCC6285 (11.1 × 10

2/mL) strain.

Further, the results were indicated that Yeast extract and malt extract media

were moderately enhanced the growth. In contrast, the growth was

significantly low in glycerol asparagines (1.4 ×102/ mL of sample) medium.

4.2.3 Effect of different Carbon Sources on the Growth of

S. sannanensis strains

Carbon sources covering different monosaccharides, disaccharides

and polysaccharides on the growth of S. sannanensis strains were carried out

to identify the array of utilization of various carbon sources. Among the

various carbon sources tested, monosaccharides followed by disaccharides

were preferred by S. sannanensis strains rather than polysaccharides for their

maximal growth (Table 4.11). The choice of carbon sources varied from

strain to strain at minimum level. The results revealed that S. sannanensis

strains (Yer11 and Kol44) preferred monosaccharides especially glucose as

the sole carbon source (12.9 × 102/mL and 12.2 × 10

2/mL of sample). The

trend remained the same with all the strains with glucose as a best carbon

source. In addition to glucose source, starch also favoured the growth of

S. sannanensis. However, fructose (11.0 × 102/mL of sample), maltose

65

(7.1 × 102/mL of sample) and sucrose (6.2 × 10

2/mL of sample) were

moderately improved the growth of S. sannanensis strains, while CMC and

pectin were found to be poor carbon sources in terms of enhancing growth.

4.2.4 Effect of various Nitrogen Compounds on the Growth of S.

sannanensis Strains

Various nitrogen sources including organic, inorganic, nitrate,

nitrogen and ammonical derivatives on the growth of S. sannanensis strains

were studied. The results revealed that S. sannanensis strains were capable of

utilizing a wide range of nitrogenous compounds. However, its efficiency in

utilizing various nitrogen sources significantly varied in varying levels

(Table 4.12). The growth of Yer11 strain was shown as 12.6 × 102/mL of

sample in ammonium sulphate followed by ammonium nitrate (9.6 × 102/mL

of sample). The organic and inorganic nitrogen sources were supporting the

growth of all selected strains partially while that of at least growth was

recorded (4.1 × 102/ mL of sample) with basal medium incorporated with urea

as a nitrogen source inoculated with Kol35 and Yer11 strains.

4.2.5 Influence of various Amino acids and Vitamins on the Growth

of S. sannanensis Strains

Influence of various amino acids and vitamins on the growth of

S. sannanensis strains were studied using basal medium incorporated with

wide sources of amino acids and vitamins. The results revealed that among

the ten amino acids tested, the maximal growth of Yer11 strain was found in

methionine (10.9 × 102/mL of sample) followed by alanine (9.8 × 10

2/mL of

sample) and aspartic acid (9.1 × 102/mL of sample). However, other amino

acids namely arginine and proline were poorly supported the growth of

S. sannanensis strains (Table 4.13).

66

Similarly, the results on the influence of vitamins on the growth of

S. sannanensis strains revealed the growth was maximal with biotin

(13.2×102/mL of sample) followed by Vit A (11.2×10

2/mL of sample) and

Vit B5 (10.2×102/mL of sample) sources. Vit B3 (9.6×10

2/mL of sample) and

Vit C (9.2×102/mL of sample) were moderately supported the growth

(Table 4.14).

Table 4.9 Standardization of different media for the growth and mass

multiplication of S. sannanensis strains

S.

sannanensis

strains

Population density of S. sannanensis strains (×102/mL)

Starch

casein

nitrate

Yeast

extract

Oat

meal

Malt

extract

Glycerol

asparagines

Yer11 9.4 4.8 1.9 2.4 1.4

Yer28 12.5 9.6 2.2 3.9 1.7

Kol35 8.2 5.4 2.2 2.6 2.0

Kol44 9.9 6.6 1.7 2.1 2.4

MTCC6285 11.1 9.3 3.2 5.1 1.9

SE± 0.82 0.71 0.62 0.43 0.34

CD P=0. 05 1.45 1.68 1.34 0.92 0.58

67

Table 4.10 Effect of different carbon sources on the growth of


Carbon

sources


Yer11 Yer28 Kol35 Kol44MTCC

6285SE±

CD

P=0. 05

Glucose 12.9 11.2 12.1 12.2 11.0 3.6 7.1

Fructose 11.0 9.8 10.8 11.6 10.2 2.8 3.9

Sucrose 6.2 5.9 4.5 5.2 6.0 1.2 2.8

Maltose 7.1 5.3 5.8 4.2 4.9 1.8 3.7

Starch 12.4 10.5 11.6 12.7 11.3 2.9 4.6

CMC 3.2 2.5 2.0 3.0 3.6 0.6 1.8

Pectin 4.2 5.1 4.6 5.5 5.9 1.1 2.6

Control 0.1 0.1 0.2 0.1 0.3 0.01 0.03

Values are the population of S. sannanensis strains at 102/ mL of sample

All the data represented in the table were the average of three replicates.

68

Table 4.11 Effect of different nitrogen sources on the growth of


Nitrogen

sources


Yer 11 Yer 28 Kol 35 Kol 44MTCC-

6285SE±

CD

P=0.05

Ammonium

sulphate12.6 9.2 12.0 9.4 10.9 2.8 5.1

Ammonium

nitrate9.6 7.1 8.6 6.8 9.1 1.6 3.6

Ammonium

chloride6.2 6.5 7.5 6.8 7.7 1.7 3.3

Sodium

nitrate7.4 5.2 6.9 5.1 6.8 1.9 3.0

Yeast

extract9.1 8.6 8.9 8.0 7.8 2.0 4.1

Potassium

nitrate5.6 4.8 6.0 5.2 5.7 1.0 2.3

Urea 4.2 5.7 4.1 5.0 5.3 0.9 1.9

Control 0.2 0.1 0.3 0.1 0.2 0.1 0.2

69

Table 4.12 Influence of various amino acids on the growth of


Amino acids

Population density of S. sannanensis strains (× 102/mL)


6285SE± CD P=0.05

Alanine 9.8 9.0 10.0 8.6 11.6 2.6 3.5

Serine 8.7 9.0 9.6 7.2 10.3 2.1 4.6

Methionine 10.9 7.4 9.0 5.1 10.6 2.0 4.3

Aspartic acid 9.1 6.2 8.6 6.8 9.8 1.5 3.5

Glutamic acid 9.0 5.2 7.2 4.2 9.2 1.3 2.8

Asparagine 8.9 5.0 5.1 7.3 9.0 1.1 2.6

Arginine 5.2 6.0 5.6 6.9 5.8 1.2 2.8

Proline 6.1 4.6 6.8 6.1 6.7 1.0 2.1

Control 0.2 0.1 0.1 0.3 0.2 0.1 0.2

70

Table 4.13 Influence of different vitamins on the growth of


Vitamins

Population density of S. sannanensis strains

(×102/mL)


-6285SE±

CD

P=0.05

Vit A 11.2 9.6 10.2 7.2 11.6 2.2 4.6

Vit B1 10.1 9.0 8.1 6.9 10.5 1.8 3.8

Vit B2 5.5 6.2 5.4 6.2 5.6 1.6 3.5

Vit B3 9.6 8.7 9.0 9.2 9.4 1.9 3.9

B5(Nicotinic

acid)10.2 9.2 7.8 10.0 11.2 2.1 4.6

Vit B7 13.2 11.3 10.9 11.0 12.6 2.4 4.9

B9 (Folic acid) 5.8 5.7 5.6 6.4 6.2 1.4 2.9

Vit C 9.2 8.8 7.2 6.7 9.9 1.2 2.6

Control 0.2 0.3 0.2 0.3 0.1 0.2 0.3

Values are the population of S. sannanensis strains at 102/ mL of sample

All the data represented in the table were the average of three replicates.

71

4.2.6 Enzymatic screening of S. sannanensis strains

The active isolates of S. sannanensis obtained from the rhizosphere

region of the Yercaud and Kolli hills were subjected to screen for the

enzymatic activity as a secondary screening method. On the other hand, the

effect of various biotic and abiotic factors on the growth metabolism of S.

sannanensis strains were studied which were considered as the primary

screening traits. It was observed that all the strains of S. sannanensis were

found to possess amylase, protease, urease, cellulase and lipase. In contrast,

chitinase was observed to be absent in all the strains (Table 4.14). In the case

of amylase and cellulase, a clear zone against dark blue and dark brown

background was registered in Starch casein agar and Czepeck mineral salt

agar; respectively by all the strains. Similarly, appearance of orange

fluorescent colonies was observed in Rhodamine B agar plates during lipase

production (Table 4.15).

All the five strains of S. sannanensis were subjected to the

secondary screening process through colorimetric analysis. The secondary

screening technique was not performed for the chitinase activity as it showed

negative result by all the five strains used. In the case of amylase, the activity

was found to be 18.0, 16.7, 22.5, 20.1 and 19.9 U/ mL by Yer11, Yer28,

Kol35, Kol44 and MTCC6285 strains; respectively. The maximum activity

was observed with Kol35 strain and minimum with Yer28 strain. Similarly,

the maximum activity of protease was observed to be 18.8 U/ mL by Kol35

strain and minimum was recorded as 9.7 U/ mL by Kol44 strain. In the case

of urease activity, 18.4 U/ mL was noted by Yer11 strain and the lowest

activity was observed as 13.6 U/ mL by Kol44 strain (Table 4.16).

72

The absorbance range of cellulase activity was totally different

when compared with the other three enzymes assayed with respect to various

S. sannanensis strains. Because the maximum activity was possessed by

MTCC6285 (23.8) strain followed by indigenous Kol35 (22.3) strain. Among

the four enzymes, the activity was found to be good with Kol35 followed by

MTCC6285 strains. The assay for the lipase activity was carried out through

titration method which showed the maximum activity was observed at 0.49,

0.38, 0.35, 0.28 and 0.31 U/ mL by Kol35, Yer11, MTCC, Kol44 and Yer28

strains; respectively (Table 4.16).

73

Table 4.14 List of enzymes involved in the enzyme screening technique by various strains of S. sannanensis

Name of the

enzymes and

substrates

Media used

Name of the strains of S. sannanensis

ObservationsYer11 Yer28 Kol35 Kol44 MTCC 6285

Amylase

(Starch)Starch casein agar ++ ++ ++ ++ ++

Clear zone against dark

blue background

Protease

(Casein)Skim milk agar ++ ++ ++ -- ++ Clear zone

Urease

(Urea)Christenson’s agar ++ -- ++ ++ ++ Appearance of pink colour

Cellulase

(CMC)

Czepeck mineral salt

agar-- ++ ++ + ++

Clear zone in dark brown

plate

Lipase

(Gingili oil) Rhodamine B agar ++ + ++ - +Appearance of orange

fluorescent colonies

Chitinase

(Chitin)Colloidal chitin agar -- --- --- -- --

No change in the medium

++Fully utilized

+Moderately utilized

--Unutilized

74

Table 4.15 Screening of enzyme activity of S. sannanensis strains by

following spectrophotometric method measuring optical

density

Name of the

strains

Optical density values (U/mL)

Amylase at

690nm

Protease at

440nm

Urease at

440nm

Cellulase at

550nm

Yer11 18.0 11.2 18.4 15.8

Yer28 16.7 12.3 17.0 16.4

Kol35 22.5 18.8 17.6 22.3

Kol44 20.1 9.7 13.6 20.1

MTCC6285 19.9 13.5 15.2 23.8

SE± 2.31 2.87 1.72 1.33

CD P=0.05 4.56 5.76 2.34 2.56

75

4.2.7 Effect of pesticide, fungicide and weedicide agrochemicals on

the growth of S. sannanensis strains

The soils are being received a huge amount of pesticides,

fungicides and weedicides in order to control a wide variety of pests and

phytopathogens under natural conditions. Various types of weedicides are

being applied in soils to control a broad spectrum of weeds covering both

monocot and dicot plants. To know the resistance behavior of S. sannanensis

strains against these agrochemicals, the present study was undertaken. The

basal medium was incorporated with various doses of these agrochemicals

and subsequently inoculated with S. sannanensis strains.

The results indicated that all the tested pesticide concentrations

inhibited the growth of S. sannanensis strains significantly when compared to

untreated control. But Yer11 strain was inhibited by propargite pesticide at

the concentration of 500 µg/mL. Other strains were not affected by this

concentration. In contrast, dicofol at 100 µg/mL concentration, Yer11 and

Kol35 strains were supporting the growth moderately. The results clearly

revealed that when concentration was increased the growth of all

S. sannanensis strains was inhibited (Table 4.16). Quinolphos was adversely

affecting the growth of S. sannanensis (0.39 OD) strains. A least resistance of

S. sannanensis strains towards pesticides (Dicofol) was recorded with

MTCC6285 strain. Among the five strains, Yer11 and Kol35 strains were

tolerated the concentration of pesticides used.

The growth of S. sannanensis strains were found to be least with

fungicides incorporated medium when compared to that of untreated control.

When the concentration of hexaconazole, copper oxychloride and

carbendazim fungicides increased, the growth of S. sannanensis strains were

decreased. The results further revealed that propioconazole was adversely

76

affected the growth (0.22 OD) of Kol35 strain. Other fungicides such as

carbendazim, mancozeb and hexaconazole at higher concentrations partially

influenced the growth of S. sannanensis strains which impart the

compatibility behaviour of S. sannanensis strains with chemical fungicides

(Table 4.17).

In order to test the resistance nature of S. sannanensis strains to

various weedicides, the basal medium was incorporated with different kinds

of weedicide at various doses and inoculated with specific strains. The results

indicated that when the concentration of Paraquat dichloride increased, the

growth of S. sannanensis strains decreased. Except Yer11 strain, other strains

were well supported at the concentration of 100 µg/mL in terms of growth.

Similar kind of results were observed in the case of 2,4-Dichlorophenoxy

acetic acid too. The maximum growth was observed with Yer28 strain (1.32

OD) at 100 µg/ mL of 2,4-Dichlorophenoxy acetic acid (Table 4.18).

77

Table 4.16 Study of pesticide resistance against S. sannanensis strains

Pesticide Dose (µg/mL) Yer11 Yer28 Kol35 Kol44 MTCC6285

SCN (Untreated) - 2.12 ±0.18 1.90 ±0.11 2.16 ±0.12 2.24±0.10 2.15±0.20

Propargite 100 0.74 ±0.01 0.60±0.02 0.72 ±0.02 0.72±0.01 0.52±0.01

300 1.00 ±0.13 0.94±0.03 0.76 ±0.07 0.81 ±0.05 0.66 ±0.02

500 0.62 ±0.04 0.91±0.05 0.84 ±0.02 1.10±0.14 0.82±0.06

Dicofol 100 0.99 ±0.05 0.34 ±0.01 0.87±0.02 0.29 ±0.02 0.30±0.01

300 0.62 ±0.02 0.71 ±0.05 0.28±0.03 0.93±0.05 0.81±0.05

500 0.28 ±0.01 0.07 ±0.03 0.09 ±0.07 0.07 ±0.01 0.02 ±0.03

Deltamethrin 100 1.27 ±0.11 0.94 ±0.04 0.72±0.05 0.89±0.05 0.81±0.02

300 0.71 ±0.04 0.52 ±0.02 0.97±0.03 0.74 ±0.04 0.57 ±0.03

500 0.29 ±0.02 0.22 ±0.01 0.01 ±0.01 0.20 ±0.02 0.21 ±0.01

Quinalphos 100 0.94±0.05 0.64 ±0.02 0.87±0.03 0.48 ±0.02 0.72 ±0.06

300 0.72 ±0.03 0.92±0.05 0.60 ±0.04 0.92±0.08 0.98±0.05

500 0.53 ±0.02 0.63 ±0.07 0.70±0.05 0.39 ±0.04 0.62 ±0.02

Fenpyroximate 100 0.97±0.04 0.77 ±0.02 0.85±0.07 0.72±0.06 0.77 ±0.01

300 0.68 ±0.05 0.95 ±0.03 0.30 ±0.08 0.40±0.030 0.98±0.08

500 0.37±0.09 0.31±0.02 0.27±0.05 0.24±0.07 0.34±0.07

Values indicate OD at 560 nm

SCN –Starch casein nitrogen medium


78

Table 4.17 Study of fungicide resistance against S. sannanensis strains

FungicideDose

(µg/mL)Yer11 Yer28 Kol35 Kol44 MTCC6285

SCN (Untreated) - 2.22 ±0.12 1.98 ±0.10 2.19 ±0.14 2.28 ±0.18 2.25 ±0.27

Hexacona

zole

100 1.12±0.11 0.94 ±0.08 0.84 ±0.08 0.79 ±0.01 0.91 ±0.04

300 0.82±0.02 0.76 ±0.02 0.64 ±0.05 0.68 ±0.02 0.78 ±0.03

500 0.57±0.05 0.31 ±0.01 0.32 ±0.04 0.39 ±0.04 0.41 ±0.05

Propicona

zole

100 1.20±0.14 1.14 ±0.13 0.87 ±0.03 1.18 ±0.15 1.42 ±0.16

300 0.97±0.07 0.96 ±0.04 0.71 ±0.01 0.98 ±0.01 0.98 ±0.05

500 0.35±0.02 0.33 ±0.02 0.22 ±0.02 0.37 ±0.02 0.34 ±0.02

Copperoxy

chloride

100 1.00±0.12 1.11 ±0.16 0.88 ±0.04 1.32 ±0.13 1.12 ±0.16

300 0.98±0.03 092 ±0.01 0.71 ±0.05 1.00 ±0.11 1.38 ±0.15

500 0.67±0.01 0.58 ±0.05 0.52 ±0.02 0.58 ±0.02 0.51 ±0.03

Mancozeb 100 0.50 ±0.01 0.88 ±0.01 0.61 ±0.02 0.82 ±0.04 0.87 ±0.04

300 0.97±0.01 0.62 ±0.04 0.91 ±0.07 0.80 ±0.02 0.67 ±0.05

500 0.66 ±0.05 0.31 ±0.02 0.90 ±0.03 0.30 ±0.04 0.31 ±0.01

Carbenda

Zim

100 0.87±0.04 0.68 ±0.01 0.81 ±0.04 0.78 ±0.06 0.82 ±0.02

300 0.61±0.03 0.91 ±0.04 0.52 ±0.02 0.88 ±0.05 0.91 ±0.03

500 0.31±0.01 0.79 ±0.05 0.31 ±0.01 0.51 ±0.02 0.52 ±0.04


SCN- Starch casein nitrogen medium


79

Table 4.18 Study of weedicide resistance against S. sannanensis strains

Weedicide Dose

(µg/mL)Yer11 Yer28 Kol35 Kol44 MTCC6285

SCN (Untreated) - 2.12 ±0.11 1.90 ±0.13 2.16 ±0.12 2.24 ±0.10 2.15 ±0.20

Paraquat dichloride 100 0.92 ±0.04 1.12 ±0.04 0.74 ±0.06 1.12 ±0.12 1.28 ±0.13

300 1.24 ±0.25 0.92 ±0.02 1.02 ±0.12 0.89 ±0.03 0.98 ±0.04

500 0.51±0.07 0.21 ±0.08 0.46 ±0.05 0.42 ±0.05 0.47 ±0.05

2,4- Dichlorophenoxy

acetic acid

100 1.30±0.14 1.32±0.17 0.91 ±0.03 1.00 ±0.12 1.12 ±0.12

300 0.82±0.05 0.89 ±0.06 0.84 ±0.07 0.89±0.04 0.96 ±0.06

500 0.59±0.03 0.52 ±0.01 0.48 ±0.02 0.48 ±0.03 0.62 ±0.01

Glyphosate

71% SG

100 0.05±0.03 0.33 ±0.04 0.19 ±0.04 0.25 ±0.07 0.33 ±0.06

300 0.40±0.08 0.52 ±0.05 0.43 ±0.05 0.36 ±0.05 0.48 ±0.04

500 0.79±0.04 0.98 ±0.01 0.61 ±0.01 0.59 ±0.01 0.61 ±0.02


SCN- Starch casein nitrogen medium


80

4.3 MOLECULAR CHARACTERIZATION OF

STREPTOMYCES SANNANENSIS STRAINS

4.3.1 Isolation of genomic DNA from S. sannanensis strains

The assessment of the quality and quantity of genomic DNA

extracted from the strains of S. sannanensis was performed based on four

different protocols and the best methodology was selected and subsequently

carried out for further studies. The results were interpreted based on the yield

of the DNA ( g/g) in wet weight of the mycelia, the purity of the DNA and

the time taken to complete the entire procedure in hours. Among the four

procedures used such as an STE loss method, CTAB method, Phenol-

chloroform extraction and commercial kit methods, the STE lysis method was

observed to show the highest yield of DNA (50-55 g/g wet weight of

mycelia) with good quality (1.83) when compared to other protocols (Table

4.20). The time taken for the process was observed to be the least in the case

of commercial kit for 1-1.5 hrs duration followed by the STE lysis method

which was about 2-3 hrs period. Phenol-chloroform extraction and CTAB

methods were found to show inferior results on all these parameters. Hence

STE lysis method was shown superior results and selected for further analysis

especially for RAPD studies.

The gel pictures of the isolated genomic DNA using different

protocols were presented in Figures 4.7-4.10. Since the molecular weight of

DNA isolated from actinomycetes would be higher the molecular weight of

the marker ranges from 0.5 kb to 23kb of Hind III digest of DNA was used

as the marker. The genomic DNA isolated from all the strains of

S. sannanensis showed a similar banding pattern which was 10kb - 21kb. In

all the extraction protocol DNA which was isolated from strains of

actinomycetes namely Yer11, Yer28, Kol35 and Kol44 was compared with

the standard strain obtained from MTCC.

81

Table 4.19 Assessment of the quality and the quantity of DNA extracted

from S. sannanensis using four different methods

S.No ResultsSTE lysis

method

CTAB

method

Phenol –

Chloroform

extraction

method

Commercial

kit method

1. Yield of genomic

DNA ( g/g) wetweight of mycelia

50 – 55 20 – 30 25 – 35 25 – 30

2. Purity of genomic

DNA1.83 1.76 1.61 1.79

3. Time taken in hrs

(Duration)2-3 3.5-4 3.5-4 1-1.5

Figure 4.7 Extraction of genomic DNA from S. sannanensis strains

using STE lysis buffer method

82

Figure 4.8 Extraction of DNA from S. sannanensis strains using CTAB

extraction method

Figure 4.9 Extraction of DNA from S. sannanensis strains using Phenol

– Chloroform extraction method

83

Lane1: Marker, Lanes2-6: Yer28, Yer11, Kol35, Kol44 and MTCC6285 strains.

Figure 4.10 Extraction of DNA from S. sannanensis strains using

Commercial kit extraction method

84

4.3.2 Random Amplified Polymorphic DNA analysis (RAPD) of

S. Sannanensis strains

The results of RAPD analysis of S. sannanensis strains performed

in order to find out the genetic relationship among the strains were tabulated

in the Table 4.21. A sum of 30 random primers was used out of which 10

primers were screened based on polymorphism. The documentation of the gel

obtained using all the 10 primers were presented in the Figure 4.11. The

results were analyzed based on the total number of bands and the percentage

polymorphism obtained by using the operon random primer. Among the

varieties of primers used, OPA10 showed 100% polymorphism followed by

the OPB09 primer which showed only 95.65% of polymorphism. The least

level of polymorphism was observed in the case of OPE05 which showed as

low as 54.54% of polymorphism. Good polymorphism was shown between all

the five strains including the standard strain obtained from MTCC,

Chandigarh, India. OPA10 and OPB09 primers were used for further

phylogenetic analysis.

Based on the presence and absence of the bands binary dataset

values were created and the Jaccard’s similarity was created. The results

showed that the similarity value between Yer11 and Kol44 strains was 0.667

which revealed that they are more similar in nature. The next similarity value

was 0.375 which showed that the Yer28 strain was similar to the standard

MTCC strain.

85

Table 4.20 List of Primers used in RAPD analysis of S. Sannanensis

strains

Name of

the

Primer

Sequence

5’ – 3’

GC

content

Total

number of

bands

Number of

Polymorphic

bands

Percentage of

Polymorphism

OPA02 TGCCGAGCTG 70 11 9 81.82

OPA09 GGGTAACGCC 70 9 7 77.78

OPA10 GTGATCGCAG 60 21 21 100.00

OPB09 TGGGGGACTC 70 23 22 95.65

OPB16 TTTGCCCGGA 60 22 21 95.45

OPB17 AGGGAACGAG 60 12 9 75.00

OPE04 GTGACATGCC 60 8 7 81.82

OPE05 TCAGGGAGGT 60 11 6 54.54

OPF06 GGGAATTCGG 70 9 6 66.67

OPG07 GAACCTGCGG 70 10 7 70.00

86

Lane1: Marker, Lanes2-6: Yer28, Yer11, Kol35, Kol44 and MTCC6285

strains.

Figure 4.11 Representation of ethidium bromide-stained 1.5% agarose

gel separation of RAPD reaction products obtained using

ten primers, OPA02, OPA09, OPA10, OPB09, OPB16,

OPB17, OPE04, OPE05, OPF06 and OPG07 with S.

sannanensis strains

87

Table 4.21 Jaccard’s similarity coefficient of S. sannanensis strains

including MTCC strain using OPA10

MTCC Yer11 Yer28 Ko135 Ko144

MTCC 1 0.375 0.333 0.111 0.250

Yer11 1 0.100 0.125 0.000

Yer28 1 0.000 0.667

Kol35 1 0.000

Kol44 1

The Cophenetic Correlation Coefficient (CP) = 0.84. The Similarity

Coefficient was represented in the Table 4.22.

The dendrogram analysis of S. sannanensis strains were created

using the similarity index for the OPA10 primer which showed the same

results where two clusters were observed. Kol35 strain was found to be

genetically unique when compared to other strains; whereas, Yer11, MTCC,

Yer28 and Kol44 strains were observed under a same clad.

The results obtained using the other two primers such as OPB09

and OPB16 showed no significant relationship among strains of S.

sannanensis. The Jaccard’s similarity index value was observed to be less

than 0.4 and hence no clear phylogenetic relationship can be predicted using

these primers. Therefore OPA10 primer was observed to be the best random

operon primer for the RAPD analysis of S. sannanensis strains.

88

Bootstrap values are indicated on the branches of the tree, distance scale is

indicated at the top.

Figure 4.12 Dendrogram analysis illustrating the genetic relationship

among S. sannanensis strains generated by UPGMA cluster

tree method


including MTCC strain using OPB09

MTCC Yer11 Yer28 Kol35 Kol44

MTCC 1 0.077 0.167 0.100 0.167

Yr11 1 0.231 0.182 0.231

Yr28 1 0.300 0.231

Ko35 1 0.182

Ko44 1



89


including MTCC strain using OPB16

MTCC Yer11 Yer28 Kol35 Kol44

MTCC 1 0.100 0.000 0.182 0.100

Yr11 1 0.091 0.000 0.000

Yr28 1 0.000 0.200

Ko35 1 0.000

Ko44 1



4.3.3 Phylogenetic unrooted tree of S. sannanensis strains

The genomic DNA of S. sannanensis strains were sequenced and

the sequence was represented in the Figure 4.13 and the details of sequencing

were mentioned below. These four sequences were used to understand the

phylogenetic relationship between the already available genomic sequences

present in the NCBI database (Maryland, USA). The Figure 4.13 showed the

position of the isolated strains such as Yer11, Yer28, Kol35 and Kol44 of

S. sannanensis. The results showed that Kol35 and Yer11 strains were found

to be very closely related to the tree and the results of which was in line with

the result obtained using Jaccard’s similarity value obtained from RAPD data.

All these results inferred that all the four strains obtained from Yercaud and

Kolli hills of eastern Ghats located in Tamil Nadu State, India belong to

S. sannanensis and they are closely related in terms of their genetic makeup.

90

>Kol35_Streptomyces_spp.

TACACATGCAAGTCGAACGATGAACCACTTAGGTGGGGATTAGTGCGAA

CGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCTGGGACAAGCCCT

GGAAACGGGGTCTAATACCGGATACTGATCCTCTTGGCATCCTGGATGA

TCGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCCTATCAGCTAGTTG

GTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGG

GCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG

CAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCTGATGCAGCGACGC

CGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGAA

GAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCCGGCTAACTACGTG

CCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTGTCCGGAATTATTG

GGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTTGTGAAAGCCCGG

GGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCTAGAGTTCGGGT

AGGGGAGATCGGAATTCCTGGGTGTAGCGGTGAAATGCGCAGATATCAG

GAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATACTGACGCTGA

GGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCA

CGCCGTAAACGGTGGGCACTAGGTGTGGGCGACATTCCACGTCGTCCGT

GCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGG

CTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATG

TGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCTTGACATACA

CCGGAAAGCATCAGAGATGGTGCCCCCCTTGTGGTCGGTGTACAGGTGG

TGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA

ACGAGCGCAACCCTTGTCCCGTGTTGCCAGCAGGCCCTTGTGGTGCTGGG

GACTCACGGGAGACCGCCGGGGTCAACTCGGAGGAAGGTGGGGACGAC

GTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCACACGTGCTACAATGG

CCGGTACAATGAGCTGCGATACCGCGAGGTGGAGCGAATCTCAAAAAGC

CGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAG

TCGCTAGTAATCGCAGATCAGCATTGCTGCGGTGAATACGTTCCCGGGCC

TTGTACACACCGCCCGTCACGTCACGAAAGTCGGTAACACCCGAAGCCG

GTGG

91

>Kol44_Streptomyces_spp.

ATTAGTGGCGAACGGGTGAGTAACACGTGGGCAATCTGCCCTGCACTCT

GGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACTGATCCTCTTGG

GCATCCTGGATGATCGAAAGCTCCGGCGGTGCAGGATGAGCCCGCGGCC

TATCAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGC

CGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGA

CTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAAGCCT

GATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTC

TTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCGCC

GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCGAGCGTTG

TCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCACGTCGGTT

GTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGGGCAGGCT

AGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGAAATGCGC

AGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGGGCCGATAC

TGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCT

GGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCGACATTCCAC

GTCGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACG

GCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG

CGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGCT

TGACATACACCGGAAAGCATCAGAGATGGTG

92

>Yer11_Streptomyces_spp.

ACCACCGGCTTCGGGTGTTACCGACTTTCGTGACGTGACGGGCGGTGTGT

ACAAGGCCCGGGAACGTATTCACCGCAGCAATGCTGATCTGCGATTACT

AGCGACTCCGACTTCATGGGGTCGAGTTGCAGACCCCAATCCGAACTGA

GACCGGCTTTTTGAGATTCGCTCCACCTCGCGGTATCGCAGCTCATTGTA

CCGGCCATTGTAGCACGTGTGCAGCCCAAGACATAAGGGGCATGATGAC

TTGACGTCGTCCCCACCTTCCTCCGAGTTGACCCCGGCGGTCTCCCGTGA

GTCCCCAGCACCACAAGGGCCTGCTGGCAACACGGGACAAGGGTTGCGC

TCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAGCC

ATGCACCACCTGTACACCGACCACAAGGGGGGCACCATCTCTGATGCTT

TCCGGTGTATGTCAAGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAA

GCCACATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTT

AGCCTTGCGGCCGTACTCCCCAGGCGGGGCACTTAATGCGTTAGCTGCG

GCACGGACGACGTGGAATGTCGCCCACACCTAGTGCCCACCGTTTACGG

CGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCC

TCAGCGTCAGTATCGGCCCAGAGATCCGCCTTCGCCACCGGTGTTCCTCC

TGATATCTGCGCATTTCACCGCTACACCCAGGAATTCCGATCTCCCCTAC

CCGAACTCTAGCCTGCCCGTATCGACTG

93

>Yer28_Streptomyces_sp.

CACTCTGGGACAAGCCCTGGAAACGGGGTCTAATACCGGATACTGATCC

TCTTGGGCATCCTGGATGATCGAAAGCTCCGGCGGTGCAGGATGAGCCC

GCGGCCTATCAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGACG

GGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGG

CCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGA

AAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTA

AACCTCTTTCAGCAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGA

AGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCGA

GCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGCTTGTCAC

GTCGGTTGTGAAAGCCCGGGGCTTAACCCCGGGTCTGCAGTCGATACGG

GCAGGCTAGAGTTCGGTAGGGGAGATCGGAATTCCTGGTGTAGCGGTGA

AATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGATCTCTGG

GCCGATACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTA

GATACCCTGGTAGTCCACGCCGTAAACGGTGGGCACTAGGTGTGGGCGA

CATTCCACGTCGTCCGTGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGG

GAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCA

CAAGCGGCGGAGCATGTGGCTTAATTCGACGCAACGCGAAGAACCTTAC

CAAGGCTTGACATACACCGGAAAGCATCAGAGATGGTGCCCCCTTGTGG

TCGGTGTACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTT

Details of 16s rDNA sequence analysis of S. sannanensis strains

94

Figure 4.13 Representation of unrooted phylogenetic relationship of S.

sannanensis strains

Yer28 strain

Yer11 strain

Kol35 strain

Kol44 strain

MTCC6285 strain

Strains isolated from Yercaud hills

Strains isolated from Kolli hills

A standard strainobtained from MTCC

95

The 16Sr DNA sequence data reveals that, all the 4 strains of

actinomycetes have unique sequences which are presented in Figure 4.13.

Based on the sequence data, the strains were identified as Streptomyces sp.

Each of the strains were submitted and the accession numbers were

Table 4.24 Accession number of the four strains isolated from Yercaud

and Kolli hills of Eastern Ghats

S.No Isolate name Accession number

1. Yer 11 KF027207

2. Yer28 KF027208

3. Kol35 KF027209

4. Kol44 KF027210

4.3.4 Extraction and expression of total proteins from S. sannanensis

strains

The amount of total proteins exacted from S. sannanensis strains

were standardized using three different protein extraction protocols such as

SDS-PAGE, TCA and Urea. The quantitative determination of proteins from

all the strains of S. sannanensis was carried out by Lowry et al (1951)

method. The estimation revealed that the specific amount of proteins was

yielded at the maximum through SDS-PAGE protocol when compared to

TCA and urea protocols. The yield of proteins was found to be of 8.6 mg,

8.2 mg, 8.5 mg, 8.8 mg and 8.6 mg with SDS-PAGE extraction method

whereas the TCA extraction yielded 6.2 mg, 5.5 mg, 6.0 mg, 6.2 mg and

5.8 mg followed by 5.5 mg, 6.0 mg, 6.4 mg, 4.5 mg and 5.0 mg of proteins

with urea extraction protocol with respect to different strains of

S. sannanensis.

96

The proteins obtained from the quantitative method were subjected

to the three protocols for the separation. The extracted proteins were subjected

to separate them based on molecular weight using SDS-PAGE electrophoresis

method. The results indicated that a total of 22 prominent bands were

recorded in SDS-PAGE method (Figure 4.14). In the case of urea extraction

protocol there was an interference of carbohydrates that resulted in the poor

yield of total proteins (Figure 4.15). Further the extraction of total proteins

was carried out by means of TCA method, where the banding pattern was

found to be unclear with failure to detect many of satellite proteins

(Figure 4.16).

The results on protein extraction profile of S. sannanensis strains

using different extraction methods for total proteins revealed that the protein

bands were found to be prominent and very clear with SDS buffer extraction

protocol followed by urea and TCA protocols (Figures 4.14 - 4.16). In the

case of TCA protocol, the number of protein bands was found to be lesser and

however common unique bands were observed especially at 31, 45 and

66 kDa molecular weight.

Figure 4.14 Extraction of total proteins from S. sannanensis strains

through SDS – PAGE protocol

97


through urea extraction protocol

Lane1: Marker, Lanes2-6: Yer28, Yer11, Kol35, Kol44 and MTCC6285 strains.


through TCA protocol

98

4.4 EFFECT OF BIOACTIVE SECONDARY METABOLITES

OF STREPTOMYCES SANNANENSIS AGAINST HUMAN

BACTERIAL PATHOGENS

The effect of crude extracts obtained from the different strains of

S. sannanensis which were isolated from Yercaud and Kolli hills were

subjected to evaluate for their antibacterial activity against the human

bacterial pathogens. The antibacterial activity was evaluated by following

three different protocols such as a Conventional streaking method, Minimum

Inhibitory Concentration Level and Well diffusion assays.

4.4.1 Antibacterial activity S. sannanensis strains through

conventional streaking method

The results on antibacterial sensitivity of S. sannanensis strains

against the human bacterial pathogens were represented in the Figures 4.17

and 4.18 by following the conventional streaking method. The results were

categorized from a minimum inhibition range of 2.4 mm to the maximum

inhibition of 5.7 mm. It was very interesting to notice that among the selected

pathogens such as Escherichia coli, Serratia marcescens, Proteus vulgaris,

Salmonella paratyphi A and Pseudomonas aeuroginosa showed a least

resistance against all the strains of S. sannanensis.

On other hand, remaining pathogens such as Bacillus subtilis,

Salmonella paratyphi B, Klebsiella pneumoniae, Staphylococcus aureus and

Clostridium spp. showed the highest resistance against all the strains of

S.sannanensis. It was noticed that Kol35 strain showed the maximum

antibacterial sensitivity of 5.7mm against Escherichia coli followed by

Serratia marcescens which accounted 5.5mm of inhibition rate. Moreover,

statically significant difference was observed between Kol35 strain and other

strains at a minimum level (Figures 4.17 and 4.18).

99

The standard culture obtained from MTCC showed poor results in

terms of a minimum zone of inhibition against Klebsiella pneumonia

(2.4mm), Salmonella paratyphi B (2.5mm) and Staphylococcus aureus

(2.5mm). Similarly, it was able to produce a least prominent inhibitory zone

was about 3.5mm against Escherichia coli, Serratia marcescens and

Clostridium spp. which was found to lower when compared with indigenous

isolates. Yer11 and Yer28 strains of S. sannanensis obtained from Yercaud

soil samples in Andrographis paniculata and Cissus quadrangularis plants;

respectively. Kol35 and Kol44 strains of S. sannanensis obtained from Kolli

hills soil samples in Cissus quadrangularis and Adhatoda zeylancia plants;

respectively. MTCC – The standard culture obtained from MTCC for

comparison purpose. There was a correlation between strains isolated from

medicinal plants and antibacterial activity against human pathogenic

microorganisms in terms of inhibition potential growth of pathogenic

microorganisms.

Figure 4.17 Antibacterial sensitivity of S. sannanensis strains against

human pathogens by conventional streaking method

100

A – Kol44, B – Yer28, C – Yer 11, D – Kol35, E – MTCC

Figure 4.18 Antagonistic activity of Streptomyces strains isolated from

Yercaud and Kolli hills against human bacterial pathogens

through Conventional streaking method

A B

C D

E

101

4.4.2 Antibacterial activity S. sannanensis strains through

well-diffusion method

The results of antibacterial activity of S. sannanensis strains

determined through wel - diffusion assay was depicted in the Figure 4.19.

The maximum zone of inhibition was noticed in Kol35 strain against all the

human bacterial pathogens in which high zone of inhibition was about 42 mm

observed with Serratia marcescens, Proteus vulgaris and Pseudomonas

aeuroginosa followed by 40 mm zone of inhibition with Escherichia coli and

Salmonella paratyphi A. Yer11 strain exhibited the zone of inhibition of

37mm, 35mm and 34mm for Escherichia coli, Serratia marcescens and

Pseudomonas aeuroginosa; respectively. The results of antibacterial activity

through well diffusion assay method were directly correlated with the results

of conventional streaking method. However, there was no statically

significant difference observed between the strains except Kol35 strain which

showed superior results (Figure 4.19 – 4.22).

Figure 4.19 Evaluation of crude extracts obtained from S. sannanensis

strains against human pathogens by well-diffusion method

102

A. Serratia marscens

B. Escherichia coli

C. Proteus vulgaris



through Well Diffusion method

Yer11

Yer28

Kol35

Kol44

MTCC

Yer11 Yer28

Kol44

Kol35

MTCC

Kol35 Kol44

Yer11

Yer28

MTCC

103

D. Salmonella paratyphi A

E. Salmonella paratyphi B

F. Pseudomonas aeruginosa




Kol35

Kol44

MTCC

Yer11Yer28

Kol35 Kol44

Yer11 Yer28

MTCC

Kol35Kol44 Yer11

Yer28

MTCC

104


Yercaud and Kolli hills against Staphylococcus aureus


4.4.3 Determination of Minimum Inhibitory Concentration level

(MIC)

The results of minimum inhibitory concentration (MIC) values

clearly revealed that all the strains of S. sannanensis along with the standard

strain were varied in varying levels in terms of the zone of inhibition and

concentration of crude extracts which ranged from 10 to 20 g mL-1

. The

minimum MIC value of 12.2 mm was recorded with MTCC standard culture

at 10 g mL-1

against Serratia marcescens. It was followed by Kol44 strain

which gave 18mm and 19mm of MIC values at 10 g mL-1

against Serratia

marcescens and Bacillus subtilis; respectively. Kol35 strain recorded the

highest MIC values of 36.5mm, 38.5mm and 40.2mm at the concentration of

10, 15 and 20 g mL-1

; respectively against Salmonella paratyphi A.

However, insignificant results were observed between the strains except

Kol35 strain which showed the highest MIC values.

Kol35

Kol44

Yer11

Yer28

MTCC

10

5

Table 4.25 Evaluation of crude extracts obtained from S. sannanensis strains against human bacterial pathogens

through Minimum Inhibitory Concentration Level (MIC)

Name of the

Bacterial

Pathogens

Concentration of crude extract (ml) *

Yer11 Yer28 Kol35 Kol44 MTCC**

10 15 20 10 15 20 10 15 20 10 15 20 10 15 20

Escherichia coli 19.5 29.8 30.5 32.5 32.8 34.2 34.5 36.5 38.5 25.0 28.2 30.3 32.3 35.2 36.5

Serratiamarcescens 27.4 27.5 29.5 30.6 31.5 32.6 29.5 30.5 33.3 18.0 25.8 20.5 12.2 27.3 29.5

Bacillus subtilis 18.2 21.5 22.5 26.5 26.5 28.7 30.5 32.5 35.2 19.0 28.5 20.5 20.3 21.6 22.0

Proteus vulgaris 22.50 28.8 29.5 23.7 24.5 26.5 25.5 29.5 30.2 23.5 26.5 29.0 23.5 26.5 29.5

Salmonella paratyphi A 21.5 22.5 25.5 29.8 30.5 33.5 36.5 38.5 40.2 27.2 29.5 32.5 26.5 27.5 30.5

Salmonella paratyphi B 29.5 29.5 31.6 28.5 29.5 32.6 35.5 36.5 38.2 23.5 25.2 29.5 24.5 26.5 28.5

Klebsiellapneumoniae 26.6 27.5 28.4 27.8 28.5 30.5 34.5 35.5 36.2 21.5 24.5 27.6 22.5 24.5 28.5

Staphylococcus aureus 20.5 20.5 22.6 27.5 27.5 29.4 31.6 33.5 35.5 22.3 23.2 26.4 24.5 25.5 30.3

Pseudomonas aeuroginosa 29.5 30.2 32.5 33.5 34.5 35.5 32.3 35.2 37.5 24.5 26.2 30.8 26.6 30.5 31.5

Clostridium sp. 22.5 24.5 25.5 26.5 28.5 29.5 30.3 31.0 35.6 23.2 25.5 26.0 25.5 27.5 29.5

* Average of three replications ** Standard culture obtained from MTCC for comparison purpose.

106

4.4.4 Effect of bioactive secondary metabolites of S. sannanensis

strains against human fungal pathogens

The crude extract containing antifungal compounds extracted from

S. sannanensis strains were tested to check the antifungal activity against

human pathogenic fungal microorganisms such as Aspergillus fumigates,

Aspergillus flavus, Stachybotrys chartarum, Histoplasma capsulatum,

Pneumocystis jirovecii, Candida albicans, Scizophyllum commune and

Ustilago maydis. Different concentrations such as 2 mL, 6 mL and 10 mL of

the extract were used to record the minimum inhibitory concentration in terms

of zone of inhibition. The results indicated that different concentrations of the

crude extract showed varying levels of minimum inhibitory concentration. In

all the cases, it’s very interesting to note that the increased in concentration of

the extract reduced the growth of the fungal pathogens (Table 4.26).

In addition to that Kol35 and Yer28 strains were observed to yield a

responsive inhibitory level when compared to Kol44 and Yer11 strains. The

maximum inhibitory concentration was found to be 23.3 mm and the

minimum was 14.0 mm using 2 mL of crude extract (Table 4.26). In the case

of 6 mL of antifungal extract concentration, the maximum and minimum

MIC values were registered as 20mm and 10mm; respectively followed by

14mm and 8mm of MIC values in 10 mL of the antifungal extract

concentration. In all the cases, MTCC strain was observed to possess a very

good antifungal activity as that of Kol35 strain.

Of all the five strains tested, Kol35 strain was found to exhibit a

reasonable antifungal activity followed by Yer28 strain. Among the human

pathogenic fungal microorganisms such as Aspergillus fumigatus Aspergillus

flavus Stachybotrys chartarum, Histoplasma capsulatum, Pneumocystis

jirovecii, Candida albicans, Scizophyllum commune and Ustilago maydis

10

7

Table 4.26 Antifungal activity of S. sannanensis strains against human pathogenic fungal microorganisms

Fungal species

Diameter of the fungal growth (mm)

Extract of Yer11

strain

Extract of Yer28

strain

Extract of

Kol35 strain

Extract of Kol44

strain

Extract of

MTCC strain

2 mL 6 mL 10 mL 2 mL 6 mL 10 mL 2 mL 6 mL 10 mL 2 mL 6 mL 10 mL 2 mL 6 mL 10 mL

Aspergillusfumigatus

17.3 13.5 10.7 20.3 17.5 13.3 23.3 20.3 14.5 19.0 16.5 12.0 20.5 17.5 14.0

Aspergillus flavus 18.3 15.0 11.5 19.3 17.7 12.3 22.5 18.5 12.5 17.7 14.5 10.0 21.7 15.5 11.5

Stachybotrys

chartarum15.0 10.0 9.0 16.5 11.0 10.5 19.7 13.7 11.5 13.3 10.3 8.5 15.7 12.7 11.3

Histoplasma

capsulatum14.7 11.3 8.5 16.5 12.5 10.3 16.7 13.0 11.0 14.5 11.0 8.7 15.5 10.7 11.5

Pneumocystis

jirovecii13.3 12.3 10.5 16.0 17.5 13.3 23.3 20.0 12.5 15.0 16.5 12.0 18.5 15.0 14.0

Candida albicans 14.0 10.5 9.7 17.7 17.7 10.3 22.5 18.5 12.5 17.7 14.5 10.0 19.7 15.5 13.5

Scizophyllum

commune17.7 12.3 9.0 18.0 12.0 10.5 19.7 17.7 11.5 14.3 13.3 9.5 15.3 12.7 12.7

Ustilago maydis15.5 10.7 8.7 16.0 12.5 11.0 16.7 15.5 11.7 14.0 13.0 9.7 16.5 11.7 12.5

108

tested, Aspergillus fumigates and Histoplasma capsulatum were registered the

highest antifungal activity at 10 mL concentration followed by 6 mL

concentration.

4.5 ANTICANCEROUS ACTIVITY OF BIOACTIVE

SECONDARY METABOLITES OF STREPTOMYCES

SANNANENSIS STRAINS

The crude extracts obtained from S. sannanensis strains such as

Yer11, Yer28, Kol35, Kol44 and MTCC were treated with human breast cancer

cell lines viz., SKBR3, MCF7 and MDA-MB231. The growth inhibition

percentage of all the three cell lines by the crude extract was shown in the

Figures 4.20 – 4.22. The results clearly indicated that inhibition of cell growth

of SKBR3 cell line by the bioactive metabolites from Kol35 and Yer28 strains

were found to be comparatively higher than Yer11 and Kol44 strains. It

revealed that the bioactive compounds obtained from Kol35 and Yer28 strains

was observed to be more potent in inhibiting SKBR3 cell growth when

compared to other bioactive metabolites obtained from Yer11 and Kol44

strains. However, bioactive metabolites extracted from Kol35 strain gave

average results in inhibiting the cell growth when compared with standard

Nolvadex drug.

Application of increased concentration of the crude extract had

significantly increased the percentage of cell growth inhibition of SKBR3 cell

line. The secondary metabolite concentration at 20 µL obtained from Kol35

strain showed 68% of growth inhibition and Yer28 strain showed 64% growth

inhibition (Figure 4.23). Similarly, the inhibition of cell growth at the lowest

concentration of 5 µL crude extract from Kol35 and Yer28 strains registered

only 29% inhibition, whereas, Yer11 and Kol44 strains showed the minimum

inhibition of 12% of growth. The activity of the standard MTCC strain showed

109

an intermediary activity between Kol35 and Yer28 strains. There was no

significant difference between strains in terms of percentage of growth

inhibition.

In the case of MCF7 cell line (Figure 4.24), the percentage of cell

growth inhibition was found to be maximal in the extract of Kol35 and Yer28

strains which showed 62% and 58%; respectively. The minimum percentage of

growth inhibition was observed in Yer11 and Kol44 strains as 25% and 38%;

respectively. Hence the inhibition of cell growth of MTCC strain showed

similar activity as that of Kol35 strain. In all the cases, it’s very interesting to

note that the increased in concentration of the extract reduced the growth of

cell lines. The results further indicated that different concentrations of the crude

extract showed varying levels of growth inhibition. However, there was no

statically significant difference observed between the strains except Kol35

strain which showed superior results in terms of growth inhibition. There was a

positive correlation between the percentage of growth inhibition of cell lines

and increasing the concentration of crude extract obtained from S. sannanensis

strains.

The maximum percentage of cell growth inhibition against

MDA-MB-231 was observed at 65% and 60% of Kol35 and Yer28 strains

(Figure 4.25) and the minimum was found to be 10% by Kol44 strain. In this

case, the inhibition of crude extract from MTCC strain exhibited an inferior

activity when compared to Kol35 strain. Interestingly, Kol35 strain showed the

maximum percentage of inhibition of cell growth in all the three human breast

cancer cell lines. Highly favorable percentage of inhibition of 65% in SKBR3

and MDA-MB231 followed by 62% inhibition in MCF7 cell line were

observed. On the other hand, the application of standard Nolvadex drug gave

significant results when compared to all the crude extracts tested. However, in

all cases the average percentage of cell growth inhibition was found to be the

110

dose dependent in nature. Increasing in concentration of crude extract increased

the percentage of cell growth inhibition in all the strains and cell lines.

Figure 4.23 Effect of crude extracts obtained from S. sannanensis strains

on the cell growth inhibition percentage of SKBR3 cell line


on the cell growth inhibition percentage of MCF7 cell line

0

20

40

60

80

100

Yer11 Yer28 Kol35 Kol44 MTCC Nolvadex

+ve

control

Pe

rce

nta

ge

of

inh

ibit

ion

Strain number

5µl 10µl 15µl 20µl

0

10

20

30

40

50

60

70

80

90


+ve

control

Pe

rce

nta

ge

of

inh

ibit

ion

Strain number


111


on the cell growth inhibition percentage of MDA-MB 231 cell

line

0

20

40

60

80

100


+ve

control

Pe

rce

nta

ge

of

inh

ibit

ion

Strain number


112

MCF 7

SKBR 3

MDA MB231

Figure 4.26 Anticancerous activity of the diverse strain Kol 35 from Kolli

hills using 3 different breast cancer cell lines.

113

4.6 MASS PRODUCTION AND ANALYSIS OF THE CRUDE

EXTRACTS FROM S. SANNANENSIS (KOL35 STRAIN)

THROUGH GC-MS ANALYSIS

The crude extract extracted from the most promising strain of

S. sannanensis (Kol35 strain) was subjected to the column chromatography to

get different fractions. The partially purified fifth fraction of the bioactive

compounds was further analyzed by GC-MS instrument. There were three

prominent peaks with retention time of 7.99 (C1), 8.85 (C2) and 10.07 (C3)

min, which suggested 32.11, 79.09 and 136.19 of molecular weights,

respectively. By using available library data, C1, C2 and C3 were determined

as silane and pyridine (7.99 min retention time), 2, 4,6-trimethyl, amino

malonic acid and 4-benzoxazin (8.85) and Tris methyl and cyclohexy

dimethoxy methyl (10.07) compounds, respectively (Figure 4.27).

Figure 4.27 GC-MS analysis of culture broth of S. sannanensis (Kol35

strain) extracted by using ethyl acetate

114

4.7 BIOSYNTHESIS OF NANOPARTICLES USING

STREPTOMYCES SANNANENSIS ISOLATED FROM

YERCAUD AND KOLLI HILLS FOR BIOMEDICAL

APPLICATIONS

Biosynthesis of gold, silver and copper nanoparticles was performed

using the efficient strain of S. sannanensis. Kol35 strain was screened as an

efficient one based on the antagonistic activity of the crude extract studied

through antifungal, antibacterial and anticancerous bioassays. Kol35 strain was

treated with chloroauric acid (HAuCl4-), silver nitrate (AgNO3) and copper

sulphate (Cu (I) SO4.5H2O) as substrates for the biosynthesis of gold, silver and

copper nanoparticles; respectively.

4.7.1 UV-Visible Light Spectroscopy Observation

The characterization of the silver, gold and copper nanoparticles

synthesized was initially performed using UV-Visible light spectroscopy. The

absorption was studied at various time intervals like 24, 48 and 72 hrs. The

spectral analysis of different nanoparticles synthesized was presented in the

Figure 4.24. The peak was observed at a wavelength of 410 – 440 nm in case of

AgNO3 treated samples and 520 – 550 nm in the case of HAuCl4- treated

samples. With respect to copper sulphate treated S. sannanensis the UV

absorption peak was observed in 530 – 580 nm. Moreover, the reaction

reached stability after 72 hrs of incubation which was shown in the UV-VIS

spectra of the nanoparticle produced. Peak values were observed

correspondingly for 24, 48 and 72 hours were at 420, 430 and 450 nm;

respectively in the case of silver nanoparticles. The biotransformation was

examined by visual inspection of the biomass as well as a measurement of the

UV visible spectra from the biomass. The colour of the reaction mixture and

colours of the harvested biomass of S. sannanensis Kol35 strain using AgNO3,

115

HAuCl4- and copper sulphate solutions was tabulated (Table 4.28) after the

incubation at 24hrs, 48hrs and 72hrs.

The synthesis of silver nanoparticles was confirmed by monitoring

the formation of brownish yellow colour. UV-Vis spectroscopy is an important

technique to determine the formation and stability of metal nanoparticles in

aqueous solution. It is well known that silver nanoparticles exhibit different

colours and size due to excitation of surface plasmon resonance (SPR). The

colour change from the appearance of pink to purple colour indicates the

formation of gold nanoparticle. The surface plasmon resonance (SPR) for the

gold nanoparticle was clearly visible as a peak in the range of 540 nm. The

maximum absorption range of the copper nanoparticles was also recorded at

560nm and a sharp peak was observed only after 48 hrs. The colour change

from pale yellow to brownish colour indicates the presence of copper

nanoparticles.

Table 4.27 Colour changes of the biomass and reaction solution of

nanoparticle biosynthesis by S. sannanensis (Kol35 strain)

S.NoType of the

Nanoparticle

At zero

hours

After

24hrs

After

48hrs

After

72hrs

Colour

absorbance

(nm)

1. Silver White Yellow Brownishyellow

Brownishyellow

420

2. Gold White Pink Purple Purple 540

3. Copper White Pale

yellow

Brown Brown 560

116

Figure 4.28 UV visible spectroscopy of nanoparticles produced by

S. sannanensis (Kol35 strain). A: Silver nanoparticles; B:

Gold nanoparticles; C: Copper nanoparticles

0

0.2

0.4

0.6

0.8

1

1.2

300 350 400 450 500 550 600

72 hrs

48 hrs

24 hrs

0

0.2

0.4

0.6

0.8

1

400 450 500 550 600

24hrs

48hrs

72hrs

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

450 500 550 600 650 700 750 800

Ab

sorb

an

ce

Wavelength (nm)

24hrs

48hrs

72hrs

A

B

C

117

4.7.2 Fourier Transform Infrared (FT-IR) Spectroscopy Observation

FT-IR study determined the chemical functional groups present in

the samples and the spectral analyses of silver, gold and copper nanoparticles

synthesized were illustrated in the Figure 4.29. From the results it was observed

that more prominent bands of silver nanoparticles were observed at 1500 and

3800 cm-1

. FTIR analysis established the occurrence of the amide (I) and (II)

bands of the protein as capping and stabilizing agent on the surface of the

nanoparticles. FTIR spectrum recorded from the chloroauric acid solution after

reaction with S. sannanensis after 72 h and the bands were seen at 650, 950 and

3500 cm-1

. The proteins can bind to gold nanoparticles either through free

amine groups or cysteine residues which is stabilized by the surface-bound

proteins. The FTIR analysis of the copper nanoparticles showed the peak at

1550 and 3600 cm-1

. In this direction, it has been analysed the proteins released

into water by the S. sannanensis in terms of the number of different proteins

secreted and their molecular weights. The synthesis of the silver, gold and

copper nanoparticles did not affect the secondary structure of the protein.

The FT-IR spectrum was recorded from the potassium iodide pellet

with biosynthesized silver nanoparticle formed after 72h of incubation with S.

sannanensis Kol35 strain. Figure 4.29 showed the amide linkages between

amino acid residues in proteins giving rise to the infrared region of the

electromagnetic spectrum. The stretching vibrations of primary amines were

observed at 1500 cm-1.

The two small bands observed represents C-N stretching

vibrations of aromatic and aliphatic amines respectively. The nanoparticles

bind either through amine groups or cysteine residues in the proteins through

electrostatic attraction of negatively charged carboxylate groups. The

absorption of the copper nanoparticle in the region of 1550 cm-1

showed the

presence of C=O and the stretching vibrations in the region of 3600 cm-1

found

to possess O – H and aldehyde as a functional group.

118

Figure 4.29 FTIR analysis of nanoparticles produced by S. sannanensis

(Kol35 strain)A: Silver nanoparticles; B: Gold nanoparticles;

C: Copper nanoparticles

A

C

B

119

4.7.3 Transmission Electron Microscope of Silver, Gold and Copper

Nanoparticles of the S. sannanensis (Kol35 strain)

The transmission electron microscopy (TEM) image of the silver

nanoparticle synthesized was represented in Figure 4.30 and indicated well

dispersed particles which were found to be spherical. The average size of these

particles was approximately 30 nm. The approximate size of the gold

nanoparticle was observed to be 18 – 24nm. The gold nanoparticles were

deposited on the mycelial cell wall of S. sannanensis. The shape and size of the

colloidal copper particles were determined by transmission electron

microscopy. A strong aggregation of the copper nanoparticles was observed

which further enhances the electrostatic attraction between the particles. The

size was measured to be 32nm.

Figure 4.30 TEM observations of (A)Silver, (B) Gold and (C) Copper

nanoparticles produced by S. sannanensis(Kol35 strain).

A

B C

120

4.7.4 Assessment of antibacterial activity of silver, gold and copper

nanoparticles biosynthesized by S. sannanensis (Kol35 Strain)

The antibacterial activity of different nanoparticles biosynthesized S.

sannanensis by such as silver, gold and copper nanoparticles was carried out

based on the observations made in well diffusion method using the different

human bacterial pathogens. The results revealed that among the different

nanoparticles tested, silver nanoparticles was found to be the best in terms of

inhibition of growth of human pathogens followed by gold and copper

nanoparticles (Table 4.28). It was observed that Kol35 strain was found to

possess good antibacterial activity in silver nanoparticles against Salmonella

paratyphi A (40.3 mm), Salmonella paratyphi B (38.5 mm), Klebsiella

pneumonia (36.0 mm), Escherichia coli (34.5 mm), Pseudomonas aeuroginosa

(28.7 mm), Serratia marcescens (32.5 mm) and Staphylococcus aureus

(34.7 mm).

Silver nanoparticle has been well known for its antibacterial

properties. Hence it was noteworthy to determine the antibacterial activity of

silver nanoparticles against the above mentioned pathogenic organisms. The

antibacterial activity of gold nanoparticles showed a reduced level of inhibition

compared to that of the silver and copper nanoparticles. The diameter of the

zone of inhibition of gold nanoparticles against S. paratyphi A (38.0 mm),

S. paratyphi B (35.5 mm), K. pneumoniae (34.0 mm), E. coli (30.5 mm),

P. aeuroginosa (27.3 mm), S. marcescens (31.3 mm) and S. aureus (33.3 mm)

were recorded and found to be varied and varying levels. But comparatively the

stability of the gold nanoparticles was observed for a prolonged time compared

with the silver nanoparticles. In the case of the copper nanoparticle of the

extract from Kol35 strain showed the least level of antagonistic property

(Table 4.28).

121

There was a positive correlation between the percentage of growth

inhibition of bacterial pathogens and nanoparticles tested (Table 4.28). Further

the results were revealed the antibacterial activity of nanoparticles

biosynthesized by S. sannanensiswas found to be superior to crude extracts in

terms of growth inhibition.

Table 4.28 Effect of different nanoparticles biosynthesized by

S. sannanensis (Kol35 strain) against the human bacterial

pathogens

S.No Name of the human

pathogens

Zone of inhibition (mm) *

Silver

nanoparticles

Gold

nanoparticles

Copper

nanoparticles

1. Salmonella paratyphi A 40.3 38.0 35.3

2. Salmonella paratyphi B 38.5 35.5 30.0

3. Escherichia coli 36.0 34.0 30.0

4. Klebsiella pneumoniae 34.5 30.5 25.5

5. Pseudomonas

aeuroginosa

28.7 27.3 22.0

6. Serratia marcescens 32.5 31.3 30.0

7. Staphylococcus aureus 34.7 33.3 30.5

SE±

CD P=0.05

4.76

6.78

3.45

5.97

3.87

5.87

* Average of three replications

4.7.5 The effect of the nanoparticles biosynthesized by S. sannanensis

strains against the breast cancer cell line

The silver, gold and copper nanoparticles of S. sannanensis (Kol35

strain) were tested against the human breast cancer cell lines such as SKBR3,

MCF7 and MDA-MB231. Different concentrations of the extract nanoparticle

solutions (10 µL, 15 µL and 20 µL) were tested and the inhibition of cell

growth was observed. The results indicated that growth inhibition was the

122

highest with gold followed by silver and copper nanoparticles. There was a

positive correlation between the percentage of growth inhibition of the cell

lines and nanoparticles tested similar to that of antibacterial activities

(Table 4.29). Among the different cell lines tested, MCF7 was better than

SKBR3 and MDA-MB231 cell lines in terms of growth inhibition.

Table 4.29 Effect of different nanoparticles biosynthesized by

S. sannanensis strains on the growth inhibition of breast

cancer cell line

Name of the

cell lines

Growth inhibition

(mm) using gold

nanoparticle ( µL) *

Growth inhibition

(mm) using silver


Growth inhibition

(mm) using copper


10 15 20 10 15 20 10 15 20

SKBR3 47.5 55.5 62.3 49.5 57.0 62.3 45.5 51.5 60.3

MCF7 50.7 60.0 65.7 55.7 65.0 66.7 46.0 51.0 63.7

MDA-MB231 45.3 55.7 59.0 50.0 57.7 60.0 41.3 54.7 55.5

* Average of three replications

09 chapter

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