APPENDIX - I

MICROSCOPIC AND BIOCHEMICAL IDENTIFICATION OF

BACTERIAL ISOLATES

1. Simple Staining

The morphology and arrangement of bacterial cells were studied by simple staining.

The isolate was placed in the centre of a clean microscopic slide smeared and heat fixed. The

slide was flooded with the basic dye crystal violet for 1 min. After exposure time, the slide

was gently washed with water and examined under the microscope. The results indicating the

shape of the organisms were noted and recorded.

2. Gram Staining

The method differentiates the bacteria of similar morphology. The isolate was placed

in the centre of a clean microscopic slide. It was heat fixed into a thin smear followed by

flooding with crystal violet staining reagent for 1 min. The slide was washed gently with a

direct stream of tap water for 2 seconds. Then the slide was flooded with iodine mordant for 1

min and washed subsequently in a gentle manner with a direct stream of tap water for 2

seconds. The smear was dried by blotting with absorbent paper. After this, the smear was

immersed with the counter stain safranin for 2 min and rewashed gently with an indirect

stream of water until no colour appeared in the wash water. Then the smear was dried with

the absorbent paper and examined under the microscope. The gram positive organisms were

indicated by the development of a purple color while the gram negative organisms were pink

colour and the results were observed.

3. Motility test

The technique was to study the motility of bacteria. A clean cover glass was taken and

a thin film of vaseline was placed around the rim of the cover glass. Then, to the centre of the

cover glass was placed a loopful of log phase test culture. The cavity slide was inverted with

cavity down over the cover glass and then pressed so that the vaseline adhered to the slide.

The slide was turned carefully upside down to make the drop hanging in the cavity. The

motility of the test culture was observed by focusing the edge of the drop with a high power

under oil immersion. The results were observed and recorded.

4. Growth on Nutrient Medium

The test cultures were grown on the nutrient agar plates and their cultural

characteristics including the abundance of growth, the size and colour of the colonies were

identified as discussed in the Bergey’s Manual of Determinative Bacteriology (9th

edition)

(Holt et al., 1994).

5. Indole Test

A loop full of 24 hrs nutrient broth cultures (isolates) were inoculated separately into

a sterile 1% tryptone broth and all tubes were incubated at 35 ± 2ºC for 24 to 48 hrs. After

incubation, to each tube was added 0.5ml of the Kovacs’ reagent and mixed thoroughly. The

positive results were indicated by the formation of a dark red-colour in the top (amyl alcohol)

layer.

6. Methyl Red Test

A loop full of 24 hrs nutrient broth cultures (isolates) were inoculated separately into

a sterile MR-VP broth and all the tubes were incubated at 35 ± 2ºC for 24 to 48 hrs. After

incubation, to each tube was added 5-6 drops of methyl red solution. The positive results

were indicated by the formation of a bright red color indicating a pH of 4.2 or less which was

a positive test while the presence of a yellow color indicated negative result.

7. Voges-Proskauer Test

A loop full of 24 hrs nutrient broth cultures (isolates) were inoculated separately into

a sterile MR-VP broth and all the tubes were incubated at 35 ± 2ºC for 24 to 48 hrs. After

incubation, to each tube was added 1 ml of 40% potassium hydroxide (plus creatine) and 3 ml

of 5% solution of - naphthol in absolute ethanol. The positive results were indicated by the

development of a pink colour in 2 – 5 min. The results were noted.

8. Citrate Test

A loop full of 24 hrs nutrient broth cultures (isolates) were streaked on the Simmons’

citrate agar slopes and all tubes were incubated at 35 ± 2ºC for 24 to 48 hrs. The positive

results were indicated by the formation of a blue colour on the streak of growth indicating an

alkaline pH (pH 7.5 and above), which were recorded.

9. Gelatin Hydrolysis test

A loopfull of 24 hrs nutrient broth cultures (isolates) were inoculated separately by

simple streaking on the plate containing agar medium supplemented with 0.4% gelatin and

incubated at 35 ± 2ºC for 24 – 28 hrs. After incubation, the plates were flooded with gelatin

precipitating reagent (15% HgCl2 in 20% (vol/vol) conc.HCl). The positive results were

indicated by the development of a clear zone around the colony, which were recorded.

10. Urease Test

A loopfull of 24 hrs nutrient broth cultures (isolates) were streak inoculated on the

Christensen’s agar medium (pH 6.8 ± 1) and incubated at 35 ± 2ºC for 24 – 48 hrs. The

positive results were indicated by a change in the colour of the medium from yellow to pink.

11. H2S Production Test

The test culture was stab inoculated and streaked on the slope of the SIM agar (pH

7.3) with a straight wire and incubated at 30ºC for 24 – 48 hrs. The positive results were

indicated by the appearance of a black colour and gas due to the production of hydrogen

sulphide.

12. Catalase Test

The test culture was inoculated on to a nutrient agar slant and incubated at 35 ± 2ºC

for 24 hrs. After incubation, 1 ml of 3% hydrogen peroxide was trickled down the slant. The

positive results were indicated by observing the evolution of bubbles continuously.

13. Oxidase Test

The test culture was inoculated on a nutrient agar slant and incubated at 35 ± 2ºC for

24 hrs. After incubation, the bacterial culture was transferred on a slide and rubbed with an

Oxidase disc containing 1% tetramethyl-p-phenyl-diamine dihydrocloride. The positive

results were indicated by an intense deep purple blue color appearing within 5 – 10 sec.

14. Nitrate Reduction Test

About 5 ml of the sterile nitrate medium (pH 7) in test tube was inoculated with a

loopful of the test culture and incubated at 35ºC for 96 hrs. After incubation, to the test

culture was added 0.1 ml of the test reagent [Solution A: Dissolved 8.0g sulphanilic acid in 1

liter of acetic acid (5mol/lit). Solution B: Dissolved 5.0g of napthalamine in 1 liter of acetic

acid (5mol/lit). Then, the equal volumes of both solution A and B were mixed together to

give the test reagent]. The positive results were observed by the formation of a red colour

within few min, which indicated the presence of nitrite and the ability of the test organism to

reduce nitrate.

15. Starch Hydrolysis Test

The test culture was streaked across the centre of the sterile starch agar (pH 7) plate

and incubated at 35ºC for 24 hrs for sufficient growth. After incubation, the plates were

flooded with the iodine solution. The positive results were indicated by clear zones around

the bacterial growth and unchanged starch appeared as blue colour.

AP

PE

ND

IX -

II

B

IOC

HE

MIC

AL

CH

AR

AC

TE

RIZ

AT

ION

AN

D I

DE

NT

IFIC

AT

ION

OF

BA

CT

ER

IAL

ST

RA

INS

IS

OL

AT

ED

FR

OM

TH

E G

UT

CO

NT

EN

TS

, C

AS

T O

F E

ISE

NIA

FO

ET

IDA

AN

D U

ND

IGE

ST

ED

SO

ILS

S.N

o

Ba

cter

ial

Iso

late

s C

ult

ure

No

.

Res

ult

of

Bio

-Ch

emic

al

Tes

ts

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

1.

Esc

her

ichia

co

li

F1

, F

8,

F1

2,

F2

2,

F3

6.

M3,

M7,

M12, M

21,

M37

H4

, H

12

, H

14, H

41

, H

47

.

S2

, S

10

, S

13

, S

23

, S

32

.

C5

, C

8,

C1

2,

C27

, C

41

.

Ro

d

- +

+

-

- +

-

- -

+

- +

-

Whit

e

gli

ster

ing

2.

Kle

bsi

ella

sp.

F5

, F

11

, F

13

, F

22

, F

43

.

M3,

M9,

M12, M

17,

M25

H4

, H

24

, H

29, H

31

, H

35

.

S5

, S

12

, S

23

, S

33

, S

43

.

C6

, C

14

, C

29

, C

39

, C

42

.

Ro

d

- -

- +

/-

+

- -

+

+

+

- +

-

Sli

my

rais

ed

gro

wth

3.

Sa

lmo

nel

la sp

.

F1

1,

F1

7,

F2

9, F

34

, F

41

.

M1

2,

M1

4,

M2

9,

M3

7,

M41

H1

0,

H1

7,

H2

3, H

31

, H

39

.

S7

, S

13

, S

16

, S

29

, S

37

.

C1

1,

C1

8,

C2

7, C

33

, C

40

.

Ro

d

- -

+

- +

+

-

- +

+

-

+

-

Th

in

gra

yis

h

gro

wth

4.

Pro

teu

s s

p.

F1

0,

F1

7,

F3

9, F

41

, F

44

.

M6,

M16,

M27, M

34,

M43

H9

, H

15

, H

19, H

31

, H

36

.

S3

, S

11

, S

24

, S

31

, S

35

.

C4

, C

16

, C

27

, C

35

, C

39

Ro

d

- +

/-

+

- +

/-

+

+

+

+

+

- +

-

Th

in

spre

adin

g

gro

wth

5.

Str

epto

cocc

us

sp

.

F1

, F

13

, F

19

, F

29

, F

33

.

M5,

M14,

M21, M

27,

M36

H1

, H

11

, H

21, H

31

, H

41

.

S1

4,

S1

7,

S2

3, S

39

, S

45

.

C2

7,

C3

1,

C3

7, C

39

, C

43

.

Co

cci

+

+

+

- -

- +

+

-

- -

- -

Th

in

even

gro

wth

6.

Ba

cill

us

sp

.

F1

1,

F1

5,

F2

9, F

31

, F

40

.

M3,

M7,

M19, M

27,

M36

H7

, H

19

, H

27, H

38

, H

40

.

S7

, S

15

, S

23

, S

37

, S

42

.

C3

, C

9,

C1

4,

C25

, C

39

.

Ro

d

+

- -

+/-

-

+

+

- -

- +

+

+

Whit

e

wax

y

gro

wth

1.S

imp

le s

tain

ing

2.

Gra

m s

tain

ing

3.I

nd

ole

tes

t 4

. M

eth

yl

red

tes

t 5

.Vo

ges

- P

rosk

auer

tes

t 6

.Cit

rate

uti

liza

tio

n t

est

7.M

oti

lity

tes

t 8

. G

elat

in

Hydro

lysi

s te

st 9

.Ure

ase

test

10.

H2S

pro

duct

ion t

est

11.

Cat

alas

e te

st 1

2.

Oxid

ase

test

13.

Nit

rate

red

uct

ion t

est

14.

Sta

rch h

ydro

lysi

s te

st

15

.Gro

wth

on

nu

trie

nt

med

ium

7.

Pse

udom

onas

sp

.

F1

4,

F1

9,

F2

8, F

34

, F

43

.

M1

3,

M1

7,

M2

9,

M3

7,

M46

H8

, H

17

, H

37, H

48

, H

50

.

S1

7,

S1

2,

S3

3, S

39

, S

44

.

C1

2,

C1

9,

C2

1, C

29

, C

40

.

Ro

d

- -

- -

+

+

+

- -

+

+

+

-

Ab

un

dan

t

thin

gro

wth

8.

Sta

phyl

oco

ccus

sp.

F9

, F

22

, F

29

, F

31

, F

47

.

M3,

M11,

M19, M

27,

M36

H2

, H

13

, H

32, H

39

, H

41

.

S1

2,

S1

9,

S3

5, S

40

, S

42

.

C1

1,

C1

5,

C2

3, C

31

, C

43

.

Co

cci

+

- +

+

/-

- -

+

- -

+

- +

-

Op

aque

go

lden

gro

wth

9.

Mic

roco

ccus

sp.

F3

, F

14

, F

22

, F

29

, F

41

.

M8,

M13,

M19, M

37,

M45

H1

7,

H1

9,

H2

5, H

37

, H

42

.

S7

, S

18

, S

39

, S

47

, S

50

.

C5

, C

14

, C

26

, C

29

, C

39

.

Co

cci

+

- -

- +

+

+

+

-

- -

+/-

-

Sm

oo

th

yel

low

gro

wth

10.

En

tero

ba

cto

r s

p.

F9

, F

18

, F

33

, F

42

, F

49

.

M1

5,

M2

2,

M2

5,

M2

9,

M35

H1

3,

H1

7,

H2

9, H

31

, H

44

.

S3

, S

27

, S

29

, S

34

, S

42

.

C2

, C

29

, C

29

, C

34

, C

41

.

Ro

d

+

- -

+

+

+

- +

/-

- +

-

- -

Whit

e

gli

ster

ing

gro

wth

11.

Sh

igel

la sp

.

F2

9,

F3

8,

F4

3, F

47

, F

49

.

M5,

M12,

M35, M

39,

M45

H1

4,

H1

9,

H2

7, H

33

, H

41

.

S5

, S

22

, S

25

, S

31

, S

44

.

C4

, C

19

, C

23

, C

35

, C

45

.

Ro

d

+

+/-

+

-

- -

- -

- +

-

- -

Ev

en

gra

yis

h

gro

wth

BIO

CH

EM

ICA

L C

HA

RA

CT

ER

IZA

TIO

N A

ND

ID

EN

TIF

ICA

TIO

N O

F P

HO

SP

HA

TE

SO

LU

BIL

IZE

RS

FR

OM

TH

E G

UT

CO

NT

EN

TS

, C

AS

T O

F E

ISE

NIA

FO

ET

IDA

AN

D U

ND

IGE

ST

ED

SO

ILS

S.N

o

Ba

cter

ial

Isola

tes

Cu

ltu

re N

o.

Bio

-Ch

emic

al

Ch

ara

cter

izati

on

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

1.

Str

epto

cocc

us

sp

PS

S-

F2,

F13,

F19,

F28,

F46.

M1,

M11,

M15, M

27,

M41

H7

, H

14

, H

19, H

31

, H

43

.

S4

, S

13

, S

33

, S

37

, S

44

.

C7

, C

11

, C

16

, C

25

, C

45

.

Coc

ci

+

+

+

- -

- +

+

-

- -

- -

Th

in e

ven

gro

wth

2.

Ba

cill

us

sp

PS

B-

F5,

F11,

F13,

F22,

F43.

M5,

M12,

M15, M

27,

M45

H1

, H

14

, H

19, H

21

, H

24

.

S9

, S

17

, S

20

, S

30

, S

40

.

C9

, C

19

, C

27

, C

36

, C

49

.

Ro

d+

-

- +

\-

- +

+

-

- -

+

+

+

Wh

ite

wax

y

gro

wth

3.

Pse

ud

om

on

as

sp

PS

P-

F15,

F19,

F27,

F37,

F44.

M1

1,

M1

7,

M1

9,

M2

7,

M3

4

H1

5,

H1

8,

H2

9, H

41

, H

47

.

S9

, S

11

, S

12

, S

19

, S

47

.

C1

3,

C2

8,

C2

9, C

37

, C

44

.

Ro

d-

- -

- +

+

+

-

- +

+

+

-

Ab

undan

t th

in

gro

wth

4.

Mic

roco

ccus

sp

PS

S-

F13,

F27,

F29,

F31,

F39.

M8,

M13,

M18, M

33,

M46

H7

, H

25

, H

29, H

33

, H

46

.

S1

, S

9,

S2

2,

S3

2,

S4

2.

C2

4,

C2

6,

C2

9, C

33

, C

37

Coc

ci

+

- +

-\

+

- -

+

- -

+

- +

-

Op

aque

go

lden

gro

wth

5.

Mic

roco

ccus

sp

PS

M-

F12,

F16, F

17,

F27,

F4

9.M

3,

M1

3, M

19

, M

37

,

M4

7H

1,

H1

1,

H2

1,

H3

1,

H4

1.

S1

4,

S1

7,

S2

3, S

39

, S

45

.

C2

7,

C3

1,

C3

7, C

39

, C

43

.

Coc

ci

+

- -

- +

+

+

+

-

- -

+\-

-

Sm

oo

th

yel

low

gro

wth

6.

Sh

igel

la s

p

PS

SG

- F

13

, F

25

, F

39

, F

41

,

F4

2.M

1,

M6

, M

16

, M

26

, M

46

H5

, H

15

, H

27, H

37

, H

41

.

S4

, S

14

, S

24

, S

35

, S

44

.

C1

, C

8,

C2

4,

C35

, C

45

.

Ro

d+

+

\-

+

- -

- -

- -

+

- -

- E

ven

gra

yis

h

gro

wth

1.

Sim

ple

sta

inin

g 2

. G

ram

sta

inin

g 3

.In

do

le t

est

4. M

eth

yl

red

tes

t 5

.Vo

ges

- P

rosk

auer

tes

t 6

.Cit

rate

tes

t 7.M

oti

lity

tes

t 8. G

elat

in

Hydro

lysi

s te

st 9

.Ure

ase

test

10. H

2S

pro

du

ctio

n t

est

11

. C

atal

ase

test

12

. O

xid

ase

test

13

. N

itra

te r

edu

ctio

n t

est

14. S

tarc

h h

ydro

lysi

s te

st

15

.Gro

wth

on

nu

trie

nt

med

ium

BIO

CH

EM

ICA

L C

HA

RA

CT

ER

IZA

TIO

N A

ND

ID

EN

TIF

ICA

TIO

N O

F P

GP

R F

RO

M T

HE

GU

T C

ON

TE

NT

S,

CA

ST

OF

E,F

OE

TID

A

AN

D U

ND

IGE

ST

ED

SO

ILS

1.

S i m p l e s

tain

ing

2.

Gra

m s

tain

ing

3.I

nd

ole

tes

t 4

. M

eth

yl

red

tes

t 5

.Vo

ges

- P

rosk

auer

tes

t 6

.Cit

rate

tes

t 7

.Mo

tili

ty t

est

8.

Gel

atin

Hydro

lysi

s te

st

9.U

reas

e te

st 1

0.

H2S

pro

duct

ion t

est

11.

Cat

alas

e te

st 1

2.

Oxid

ase

test

13

. N

itra

te r

edu

ctio

n t

est

14

. S

tarc

h h

ydro

lysi

s te

st 1

5.G

row

th o

n

nu

trie

nt

med

ium

Sl.

No.

Ba

cter

ial

Iso

late

s C

ult

ure

No.

Bio

-Ch

emic

al

Ch

ara

cter

izati

on

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

1.

Azo

tob

act

er s

p.

AZ

-

F2

, F

5, F

9,

F1

8, F

46

.

M1

, M

4,

M1

5,

M2

7, M

36

H4

, H

9,

H1

7, H

42

, H

47

.

S3

, S

17

, S

32

, S

41

, S

49

.

C7

, C

21

, C

29

, C

42, C

50

Ro

d

-ve

+

+/-

+

+

M

+

+

+

+

+

+

+

F

lat,

Sli

my

, P

aste

lik

e

Co

lon

ies

2.

Azo

spir

illu

m s

p.

AG

-

F4

, F

9, F

17

, F

28

, F

36

.

M2

, M

9,

M1

5,

M1

7, M

46

H2

, H

17

, H

29

, H

44

, H

50

.

S9

, S

22

, S

37

, S

43

, S

47

.

C3

, C

11

, C

22

, C

37, C

46

.

Ro

d

-ve

_

+

+

_

M

- +

+

+

+

+

_

P

ale

yel

low

, W

hit

e

3.

Rh

izo

biu

m s

p.

RB

-

F5

, F

18

, F

37

, F

38

, F

44

.

M1

, M

7,

M1

7,

M3

1, M

45

H2

, H

13

, H

39

, H

48

, H

49

.

S1

, S

12

, S

27

, S

33

, S

37

.

C2

, C

13

, C

29

, C

33, C

48

.

Ro

d

-ve

+/-

+

+

_

M

-

+/-

+

/-

+

+

_

_

Cir

cula

r, C

on

vex

. S

emi-

tran

slu

cen

t ra

ised

an

d

mu

cila

gin

ou

s c

olo

nie

s

4.

Act

ino

myc

etes

AM

-

F1

, F

19

, F

29

, F

38

, F

49

.

M6

, M

19

, M

25

, M

37

, M

44

H1

2, H

18

, H

39

, H

41

, H

48

.

S2

, S

12

, S

23

, S

33

, S

42

.

C6

, C

13

, C

26

, C

34, C

41

.

Ro

d

+v

e _

+

/-

_

_

M

+/-

_

_

_

+

/-

+/-

+

/-

Sm

oo

th, w

hit

e co

lon

ies,

po

wd

ery c

olo

nie

s

AP

PE

ND

IX-

III

MO

RP

HO

LO

GIC

AL

ID

EN

TIF

ICA

TIO

N O

F F

UN

GA

L S

TR

AIN

S I

SO

LA

TE

D F

RO

M T

HE

CA

ST

S A

ND

GU

T R

EG

ION

S O

F E

.FO

ET

IDA

AN

D

UN

DIG

ES

TE

D S

OIL

S

S.N

o

Fu

ngal

Isola

tes

Co

lon

y C

ha

ract

eris

tics

on

So

lid

Med

ia

Mic

rosc

op

ic M

orp

ho

log

y

1.

Rh

izop

us

sp

Show

n

Rap

id

gro

wth

w

ith

vo

lum

ino

us

wh

ite

to

gre

y

aeri

al

my

celi

um

lik

e a

pep

per

ed a

pp

eara

nce

Sp

ora

ng

iosp

ore

s w

ere

lon

g,

unb

ranch

ed,

and c

lust

ered

at

nodes

opp

osi

te r

hiz

oid

s.

2.

Mu

cor

sp.

Show

n ra

pid

gro

wth

w

ith a

cott

ony

surf

ace

Sp

her

ical

Sp

ora

ng

iosp

ore

s w

ere

seen

wit

hin

th

e sp

ora

ng

ium

an

d

aro

un

d t

he

colu

mel

la (

a d

om

e- s

hap

ed s

tru

ctu

re l

oca

ted

at

the

top

end o

f th

e S

pora

ngio

spore

s).

3.

Asp

erg

illu

s sp

.

Th

e su

rfac

e co

lor

of

the

colo

ny

was

wh

ite,

b

eco

min

g g

ray

to

g

reen

w

ith

age.

Th

e su

rfac

e te

xtu

re w

as c

ott

on

y.

Co

nid

iosp

ore

s w

ere

thin

w

alle

d,

smooth

, gre

en,

and en

d in

a

hem

isp

her

ical

ves

icle

. T

he

ves

icle

was

a s

wo

llen

par

t o

f th

e ce

ll

loca

ted

at

th

e en

d.

Co

nid

ia

wer

e si

ng

le-c

elle

d,

glo

bo

se,

ech

inu

late

(co

ver

ed w

ith

sp

ines

), t

hin

-wal

led

.

4.

Pen

icil

liu

m s

p.

Show

n t

he

rap

id g

row

th w

ith y

ello

w

to g

reen

co

lor.

The

conid

iop

hore

s w

ere

erec

t, d

isti

nct

, usu

ally

bra

nch

ed,

smo

oth

to r

ou

gh

, an

d h

yal

ine

to c

olo

r. F

lask

- sh

aped

, hyal

ine

phia

lides

are

bo

rne

at t

he

apex

of

the

term

inal

met

ula

e.

5.

Ab

sid

ia s

p.

Th

e co

lon

y w

as g

ray

w

ith

a

coar

se,

wooly

tex

ture

.

Rh

izo

ids

wer

e p

rese

nt,

bu

t th

e S

pora

ngio

spore

s ra

ised

bet

wee

n

the

no

des

of

the

sto

lon

.

6.

Fusa

riu

m s

p.

Th

e co

lonie

s w

ere

flu

ffy

in

textu

re

and

var

iab

le c

olo

r.

Ob

serv

ed

the

pro

du

ctio

n

of

can

oe-

shap

ed,

mult

icel

led

mac

roco

nid

ia

and

o

ne

or

two

-cel

led

hyal

ine

mic

roco

nid

ia,

usu

ally

hel

d t

og

eth

er i

n m

ucu

s b

alls

. T

he

mac

roco

nid

ia g

ener

ally

wer

e b

orn

e in

ban

ana

like

clust

ers,

whic

h d

islo

dg

ed e

asil

y a

nd

flo

ated

fre

e fr

om

th

e h

yp

hae

.

7.

Alt

ern

ari

a s

p.

Sh

ow

n t

he

rap

id g

row

th w

ith

co

tto

ny

and g

ray t

o b

lack

. T

he

conid

iop

hore

s

wer

e d

emat

iace

ou

s re

fers

to

th

e

bro

wn

or

bla

ck c

olo

r.

Th

e co

nid

ia o

f A

lter

na

ria

sp

p.

dev

elop

ed b

ran

chin

g c

hai

ns

at t

he

apex

of

the

con

idio

ph

ore

, w

ith

the

yo

un

ges

t co

nid

ium

at

the

apex

of

each

chai

n.

8.

Tri

cho

der

ma s

p.

Sh

ow

n

rap

id

gro

wth

w

ith

w

hit

e to

gre

en i

n c

olo

r an

d t

hei

r te

xtu

re w

as

cott

on

y.

The

hyp

hae

wer

e se

pta

te a

nd b

ranch

ed.

Co

nid

iop

ho

res

wer

e sh

ort

and

bra

nch

ed w

ith

fla

sk –

ph

iali

des

. P

hia

loco

nid

ia w

ere

seen

as

clu

ster

s o

f si

ng

le-c

elle

d m

icro

con

idia

.

9.

Candid

a s

p.

The

colo

nie

s w

ere

usu

ally

sta

rk w

hit

e

colo

r, b

ut

chan

ged

to

cre

am c

olo

red

wit

h

age.

T

hey

w

ere

gla

bro

us,

crea

my

, o

r m

emb

ran

ou

s, an

d se

en a

frin

ge

of

sub

mer

ged

hyp

hae

Sh

ow

n t

he

pre

sen

ce o

f g

lob

ose

to

ov

oid

bla

sto

con

idia

an

d w

ell

dev

elo

ped

p

seu

do

hy

ph

ae

and

po

siti

ve

resu

lts

in

ger

m

tub

es

ob

serv

atio

n

10.

Sa

cch

aro

myc

es s

p.

Th

e co

lon

ies

wer

e re

sem

ble

s ca

ndid

a

sp. in

co

lor

and

tex

ture

.

Th

e ce

lls

app

eare

d a

s ovel

to s

pher

ical

, an

d m

ay e

xis

t as

eit

her

hap

loid

s o

r d

iplo

ids.

Cel

ls m

ay f

orm

sh

ort

ch

ain

s an

d e

lon

gat

e as

rud

imen

tary

pse

udp

hyp

hae

.

APPENDIX – IV

SCREENING, PRODUCTION AND ASSAY OF VARIOUS HYDROLYTIC

ENZYMES FROM VARIOUS BACTERIAL STRAINS ISOLATED FROM

EARTHWORMS GUT

1. Screening for amylase activity

The purified bacterial isolates from earthworms gut were plated on the starch

agar medium containing starch (1% w/v) and incubated at 37 ºC for 48 h. After

incubation, the plates were flooded with iodine solution. The amylolytic activity was

measured by the zone of clearance around the bacterial growth. Depending on the

clear zone of hydrolysis, the best positive organisms were selected and maintained on

nutrient agar slants at 4ºC for further experimental studies.

1. (a). Amylase Production

About 90ml of the sterile liquid medium for amylase enzyme production was

inoculated with 10 ml of Bacillus sp., Pseudomonas sp., Azotobacter sp., and

Actinomycetes individually and incubated at 37 ºC for 48 h in a shaking incubator

(150 rpm). After 48 h of incubation, the cells were harvested after a spin at 15000 rpm

for 10 min and the clear crude supernatant was stored at 4 ºC for further studies.

1. (b). Amylase Assay

Amylase is a starch degrading enzyme acting on glycogen and related

polysaccharides. - amylase causes endo-cleavage of substrates and hydrolyse 1, 4

linkages in a random manner. It has the ability to by-pass 1, 6 branch points. -

amylase hydrolyses alternate bonds from the non-reducing end of the substrate. The

enzyme degrades amylase, amylopectin or glycogen in the exo or stepwise fashion by

hydrolyzing alternate glycosidic bonds. The end product is – maltose (Bernfield,

1955).

Amylase enzyme was assayed in the reaction mixture containing 1.0 ml of

enzyme solution with 1.0 ml of starch and incubated at 37 ºC for 15 minutes in a

water bath. To this, 2.0ml of dinitrosalicyclic acid (DNS) was added to each tube for

stopping the reaction and kept in a boiling water bath for 5 minutes. The solution was

then cooled at room temperature and diluted to 10ml with distilled water. The

absorbance was read at 560nm. The absorbance values were plotted on a standard

graph drawn for D-glucose which had the concentrations ranging from 10 µg to 100

µg. The amylase activity was measured in units (U). One unit of enzyme was defined

as the amount of enzyme that released 1µmol of D-glucose/mm of the crude

extract/minute.

Calculations:

Determine the µ moles of glucose using the standard curve.

Units/ml enzyme =

Where

df= Dilution factor

10= Time of assay (in minutes) as per Unit Definition

0.1 = Volume (in milliliters) of enzyme used

2. Screening for Proteolytic Activity

The purified bacterial isolates from the earthworms gut were plated on the

Casein agar medium (casein 1% w/v) and incubated at 37 ºC for 48 hrs. The plates

were then flooded with 25% TCA (trichloro acetic acid) solution and incubated for 15

min at 45 ºC. The results were noted by measuring the zone of clearance around the

bacterial colonies on medium. Depending on the clear zone of hydrolysis, the best

positive organisms were selected and maintained on nutrient agar slants at 4 ºC for

further experimental studies.

2. (a). Protease Production

About 90ml of the sterile liquid medium for the production of protease

enzyme [glucose (1.0g/l), peptone (10.0g/l), K2HPO4 (0.5g/l), and MgSO4 (0.1g/l)]

was inoculated with 10 ml of Bacillus sp., Pseudomonas sp., Azotobacter sp., and

Actinomycetes individually and incubated at 37 ºC for 48 hrs in a shaking incubator

(150 rpm). After 48 hrs of incubation, the cells were harvested at 15,000 rpm for 10

min and the clear crude supernatant was stored at 4 ºC for further studies.

2. (b). Protease Assay

The blue colour developed by the reduction of phosphomolybdic

phosphotungstic components in the Folin-Ciocalteau reagent by the aminoacid

tyrosine present in the protein plus the blue colour developed by biuret reaction of the

protein with alkaline cupric tartrate were measured in the Lowry’s method (Ladd and

Butler, 1972).

. The protease enzyme was assayed in the reaction mixture containing 0.5ml of

the enzyme solution and 5 ml of the Tris-HCl buffered (9.0) casein and incubated at

37 ºC for 30 minutes. After incubation, the reaction was terminated by adding 5 ml of

110 mM trichloroacetic acid (TCA) and centrifuged at 10000 rpm for 5 min. The

released amino acids were measured as tyrosine using the Folin and Ciocalteau

reagent and the intensity of the colour was measured at 700 nm. The absorbance

values were plotted on the standard graph drawn with tyrosine which had the

concentrations ranging from 10 µg to 100 µg. The protease activity was measured in

units (U). One unit of the enzyme was defined as the amount of enzyme that released

1µmol of tyrosine/mm of crude extract / minute.

Calculations:

Determine the µ moles of tyrosine using the standard curve.

Units/ml enzyme =

Where

df= Dilution factor

10= Time of assay (in minutes) as per Unit Definition

0.4 = Volume (in milliliters) of enzyme used

3. Screening for Phosphatase Activity

The purified bacterial isolates from the earthworm’s gut were plated on the

Pikovskaya’s medium and incubated at 37 ºC for 48 hrs. After incubation, the

phospholytic activity was measured from the appearance of the clear zones around the

bacterial growth. The bacterial isolates representing higher phospholytic activity were

selected and maintained on nutrient agar slants at 4ºC for further experimental studies.

3. (a). Phosphatase Production

About 90ml of the sterile liquid medium for the production of phosphatase

enzyme containing Ca3(PO4)2 as the sole phosphate source was inoculated with 10 ml

of Bacillus sp., Pseudomonas sp., Azotobacter sp., and Actinomycetes inoculum

individually and incubated at 37 ºC for 48 hrs in a shaking incubator (150 rpm). After

48 hrs of incubation, the cells were harvested after centrifugation at 15,000 rpm for 10

min and the clear crude supernatant was stored at 4 ºC for further studies.

3. (b). Acid Phosphatase Assay

Acid phosphatase hydrolyses a number of phosphomonoesters and

phosphoproteins. The activity of alkaline phosphatase was estimated by the method of

Mahesh et al. (2010).

To determine the acid phosphatase activity, one ml of the culture supernatant

was incubated along with 2.0 ml of citrate buffer (pH 5.3) in a test tube containing

0.5ml magnesium acetate solution, 2.0 ml of p-nitrophenyl phosphate and made up to

7.0ml with the citrate buffer. The mixture was incubated at 37ºC for 1 hr and the

reaction was stopped by adding 2.0ml of 10% TCA. Distilled water was used in the

reaction instead of the supernatant to prepare blanks. After incubation, the control and

the test samples were centrifuged at 10,000g for 10minutes. From this, 0.5ml of the

supernatant for testing along with control were taken in test tubes and the volume in

each tube was made up to 5.0ml by adding water. To each tubes, 1.0ml of ammonium

molybdate and 0.4ml of ANSA reagent were added and mixed well. After 10

minutes, the mixture was diluted to 10.0 ml and the concentration of p-nitrophenol

was determined by measuring the intensity of blue colour at 660nm against the

reagent blank and compared with a standard curve. One unit of the enzyme activity

was defined as the amount of enzyme (mg) required for liberating 1 µ mole of p-

nitrophenol/min.

3. (c). Alkaline Phosphatase Assay

Alkaline phosphatase helps in hydrolyzing the phosphate esters, such as esters

of phenol and amines. The activity of alkaline phosphatase was estimated by the

method of Mahesh et al. (2010).

To determine the alkaline phosphatase activity, one ml of the culture

supernatant was incubated with 2.0 ml of glycine NaOH buffer (pH 9.0) in a test tube

containing 0.5ml magnesium acetate solution, and 2.0 ml of p-nitrophenyl phosphate

and made up to 7.0 ml with the glycine NaOH buffer. The mixture was incubated at

37 ºC for 1 hr and the reaction was stopped by adding 2.0ml of 10% TCA. Distilled

water was used in the reaction instead of the supernatant to prepare blanks. After

incubation, the control and the test samples were centrifuged at 10,000g for

10minutes. From this, 0.5ml of the supernatant for testing along with control were

taken in test tubes and made up to 5.0ml by adding water. To each tubes, 1.0ml of

ammonium molybdate and 0.4ml of ANSA reagent were added and mixed well.

After 10 minutes, the mixture was diluted to 10.0 ml and the concentration of p-

nitrophenol was determined by measuring the intensity of blue colour at 660nm

against the reagent blank and compared with a standard curve. One unit of the enzyme

activity was defined as the amount of enzyme (mg) required for liberating 1 µ mole of

p-nitrophenol/min.

Calculations:

Determine the 1 µ mole of p-nitrophenol/min using the standard curve.

Units/ml enzyme =

where

df= Dilution factor

10= Time of assay (in minutes) as per Unit Definition

0.5 = Volume (in milliliters) of enzyme used

4. Screening for Cellulase Activity

The purified bacterial isolates from the earthworms gut were plated on the

Carboxy methyl cellulose agar medium containing carboxy methyl cellulase (1%

w/v) and incubated at 37 ºC for 48 hrs. After incubation, the plates were flooded with

1% congored and allowed to stand for 15 min at room temperature. One molar NaCl

was thoroughly used for counter staining the plates. The cellulolytic activity was

measured by recording the appearance of clear zones around the bacterial growth. The

bacterial colonies having the largest clear zones were selected and maintained on

nutrient agar slants at 4 ºC for further experimental studies.

4. (a). Cellulase Production

About 90ml of the sterile liquid medium for cellulase enzyme production was

inoculated with 10 ml Bacillus sp., Pseudomonas sp., Azotobacter sp., and

Actinomycetes, individually and incubated at 37 ºC for 48 hrs in a shaking incubator

(150 rpm). After 48 hrs of incubation, the cells were harvested after centrifuging the

cultures at 15000 rpm for 10 min and the clear crude supernatant was stored at 4 ºC

for further studies.

4. (b). Cellulase Assay

Hydrolysis of crystalline cellulose is a complex process. Initiation of

hydrolysis of native cellulose is done by exo- 1, 4 glucanase (C1- cellulase). This

enzyme spilt alternate bonds from the non-reducing end of cellulose chain yielding

cellobiose. The endo-glucanase (Cx-cellulase) acts on carboxyl methyl cellulose. This

enzyme does not act on native cellulose. -glucosidases (cellobiase) play an important

role in the degradation of cellulose by hydrolyzing cellobiose which is an inhibitor of

exo-glucanase (Ghosh, 1987).

Cellulase enzyme was assayed in the reaction mixture containing 0.15ml of

the enzyme with 0.45ml of 1% Carboxy Methyl Cellulose (CMC) solution and

incubated at 55ºC for 15 min. The solution was again warmed for 5 min and 0.5ml of

dinitrosalicyclic acid (DNS) solution was added. To this warm solution, 1.0ml of 10%

sodium potassium tartarate solution (Rochelle salt solution) was added, the solution

was cooled at room temperature and diluted to 10 ml with distilled water. The

absorbance value was measured at 540nm. Simultaneously, a series of standards were

run using glucose with concentrations ranging from 50µg to 500µg/ml. The enzyme

activity was measured as µ moles of glucose released per minute

Calculations:

Determine the µ moles of glucose using the standard curve.

Units/ml enzyme =

Where

df= Dilution factor

10= Time of assay (in minutes) as per Unit Definition

0.2= Volume (in milliliters) of enzyme used

5. Screening for Xylanase Activity

The purified bacterial isolates from the gut of earthworm were plated on the

xylan agar medium containing xylan (1% w/v) and incubated at 37 ºC for 48 hrs.

After incubation, the plates were then flooded with congored solution (0.1% and

incubated for 15 min at room temperature followed by repeated washing with NaCl (1

M). The results were noted by measuring the zone of clearance around the bacterial

colonies on the medium. Depending on the formation of clear zone of hydrolysis, the

best positive organisms were selected and maintained on nutrient agar slants at 4ºC

for further experimental studies.

5. (a). Xylanase Production

About 90ml of the sterile liquid medium for the production of xylanase was

inoculated with 10 ml Bacillus sp., Pseudomonas sp., Azotobacter sp., and

Actinomycetes inoculum individually and incubated at 37 ºC for 48 hrs in a shaking

incubator (150 rpm) After 48 h of incubation, the cells were harvested at 15,000 rpm

for 10 min and the clear crude supernatant was stored at 4 ºC for further studies.

5. (b). Xylanase Assay

Xylanases hydrolyze xylan, a polysaccharide made of xylopyranose residues

linked by – 1, 4 glycosidic bonds (Bucht and Eriksson, 1969).

To determine the xylanase activity, 0.5 ml of the bacterial culture supernatant

was incubated with 1% xylan solution (prepared in 0.01 M citrate buffer, pH 4.8) at

50ºC for 30 min followed by the addition of 3.0ml of DNS reagent and the reducing

sugar was measured. From this, 0.5 to 3.0ml of the extract was transferred to test

tubes and each tube was equalized to a volume of 3.0ml with water. Then, 3.0ml of

DNS reagent was added and the contents were heated in a boiling water bath for 5

minutes. When the contents of the tubes were still warm, 1.0ml of 40% Rochelle salt

solution was added. The contents in the tubes were cooled and the intensity of dark

red colour was measured at 510nm. Simultaneously, the absorbance values were

plotted on the standard graph drawn for xylose with concentration ranging from

1mg/ml to 10 mg/ml. Xylanase activity was expressed as m moles/ mg of protein.

Calculations:

Determine the µ moles of xylose using the standard curve.

Units/ml enzyme =

Where

df= Dilution factor

10= Time of assay (in minutes) as per Unit Definition

0.7 = Volume (in milliliters) of enzyme used

6. Screening for Lipase Activity

The purified bacterial isolates from the earthworms gut were plated on the

tributyrin agar medium containing tributyrin (1% w/v) and incubated at 37 ºC for 48

hrs. After incubation, the lipolytic activity was measured by the appearance of clear

zones around the bacterial growth. The bacterial isolates representing higher lipolytic

activity were selected and purified on tributyrin agar plates and maintained on nutrient

agar slants at 4 ºC for further experimental studies.

6. (a). Lipase Production

About 90ml of the sterile liquid medium for lipase enzyme production was

inoculated with 10 ml Bacillus sp., Pseudomonas sp., Azotobacter sp., and

Actinomycetes of the inoculum individually and incubated at 37 ºC for 120 hrs in a

shaking incubator (150 rpm) The samples were collected after every 24 hrs and

centrifuged at 8000 rpm for 10 min and the clear crude supernatant was stored at 4 ºC

as a source of lipase for further studies.

6. (b). Lipase Assay

Lipase hydrolyses triglycerides to release free fatty acids and glycerol

(Jayaraman, 1981).

Triglycerides + H2O Glycerol + Fatty acids

Lipase activity was assayed quantitatively by using para-nitro phenyl laurate

(pNPL) as the substrate (Hasan and Hameed, 2001). About 10 ml of isopropanol

containing 30 mg pNPP was mixed with 90 ml of 0.05 M sodium phosphate buffer

(pH 8) containing 207 mg of sodium deoxycholate and 100 mg gumarabic. A total

volume of 2.4 ml of the freshly prepared substrate solution was prewarmed at 37ºC

and mixed with 0.1 ml enzyme solution. After 15 min of incubation at 37 ºC,

absorbance at 410 nm was measured against the blank. One enzyme unit (U) was

defined as the amount of enzyme required to release 1µmol of p-nitrophenol from the

substrate in milliliters per minute.

Calculations:

Determine the µ moles of p-nitrophenol using the standard curve.

Units/ml enzyme =

Where

df= Dilution factor

10= Time of assay (in minutes) as per Unit Definition

0.8 = Volume (in milliliters) of enzyme used

7. Screening for Urease Activity

The purified bacterial isolates from the earthworms gut were plated on the

Urea agar medium (Christensen medium) and incubated at 37 ºC for 48 hrs. When the

color of the medium changed into pink, it indicated the urease production by the

organism. Depending on pinkness and intensity of the color, the best positive

organisms were selected and maintained on nutrient agar slants at 4 ºC for further

experimental studies.

7. (a). Urease Production

About 90ml of sterile liquid medium for the production of urease enzyme

was inoculated with 10 ml of Bacillus sp., Pseudomonas sp., Azotobacter sp., and

Actinomycetes culture individually and incubated at 37 ºC for 48 hrs in a shaking

incubator (150 rpm). After 48 hrs of incubation, the cells were harvested after

centrifugation at 15000 rpm for 10 min and the clear crude supernatant was stored at 4

ºC for further studies (Yang et al., 2008).

7. (b). Urease Assay

Urease is present in large amounts in biologically active samples, since many

microorganisms hydrolyze urea enzymatically. Assay on urease activity gives

information on the rate of degradation of nitrogen-containing compounds especially

proteins and the activity of different fertilizers. The method described here was

developed for the examination of urease activity in plant and composted materials.

Urease catalyses the reaction

H2N- CO-NH2 + H2O CO2 + 2NH3

The urease activity was measured by phenol-hypochlorite assay

(Weatherburn, 1967). The reactions were carried out in micro tubes containing 100 µl

of the sample solution, 500 µl of 50 mM urea and 500 µl of 100 mM potassium

phosphate buffer (pH 8.0) giving a total volume of 1.1 ml. The reaction mixture was

incubated at 37 ºC for 30 min in a shaking water bath. The reaction was stopped by

transferring 50 µl of the reaction mixture to the tubes containing 500 µl of phenol-

sodium nitroprusside solution (0.05 g sodium nitroprusside and 1g phenol in 100 ml

distilled water). About 500 µl of alkaline hypochlorite (3.56 g Na2HPO4 and 1 ml

sodium hypochlorite in 100 ml distilled water) was added to the tubes, and incubated

at room temperature for 30 min. Finally, the optical density of the coloured complex

was measured at 630 nm against the blank (500 ml phenol nitroprusside sodium and

500 ml sodium hypochlorite in 50 ml distilled water) in a spectrophotometer and

compared with the standard curve prepared with (NH4)2SO4. Controls used for the

enzyme reactions were reaction mixture without the substrate and reaction mixture

without incubation. One unit of urease activity was defined as the amount of enzyme

liberating 1 mg NH3 from urea per minute.

Calculations:

Determine the 1 mg NH3 from urea using the standard curve.

Units/ml enzyme =

Where

df= Dilution factor

10= Time of assay (in minutes) as per Unit Definition

0.6 = Volume (in milliliters) of enzyme used

8.Optimization of Cultural Conditions for Enzymes Production

The optimization for the production of different enzymes employing the bacterial

isolates namely Bacillus sp., Pseudomonas sp., Azotobacter sp., and Actinomycetes

were carried out using the undercited parameters and checked for enzymatic activities

of amylase, cellulase, lipase, phosphatase, protease, urease, and xylanase.

8. (a). Effect of Incubation Period on Enzymes Production

The optimal incubation period for the production of various enzymes namely

cellulase, amylase, protease, xylanase, lipase, acid alkaline phosphatase, and urease in

the respective medium by the bacterial isolates Bacillus sp., Pseudomonas sp.,

Azotobacter sp., and Actinomycetes were carried out at different periods from 24 to

96 hrs at 37 ºC in an orbital shaker at an agitation speed of 150 rpm. Samples were

retrieved at 8 hrs intervals after centrifugation at 8000 rpm for 20 min at 4 ºC. The

culture supernatant was collected as a crude enzyme solution and assayed for different

enzymatic activities.

8. (b). Effect of pH on Enzyme Production

To check the effect of pH on the production of various enzymes namely

cellulase, amylase, protease, xylanase, lipase, acid alkaline phosphatase, and urease in

the respective medium by the bacterial isolates Bacillus sp., Pseudomonas sp.,

Azotobacter sp., and Actinomycetes, experiments were carried out at different pH

ranging from 3 to 10 at 37 ºC for 48 hrs in an orbital shaker at an agitation speed of

150 rpm. Samples were incubated at 37ºC for 48 hrs and were drawn after every 8 hrs

intervals and centrifuged at 8000 rpm for 20 min at 4 ºC. The culture supernatant thus

collected was used as the crude enzyme solution and assayed for different enzyme

activities.

8. (c). Effect of Temperature on Enzyme Production

To observe the temperature optima for the production of various enzymes

namely cellulase, amylase, protease, xylanase, lipase, acid alkaline phosphatase, and

urease in the respective medium, by the bacterial isolates Bacillus sp., Pseudomonas

sp., Azotobacter sp., and Actinomycetes, the media was inoculated and incubated at

different temperatures 20ºC, 25ºC, 30ºC, 35ºC and 40ºC for 48 hrs in an orbital shaker

at an agitation speed of 150 rpm. Samples were taken at 8 hrs intervals and

supernatant harvested by centrifugation at 8000 rpm for 20 min at 4 ºC. The

supernatant collected was used as the crude enzyme for the assay of different

enzymatic activity.

8. (d). Effect of Agitation on Enzyme Production

In order to evaluate the effect of agitation speed on the production of various

enzymes the cellulase, amylase, protease, xylanase, lipase, acid alkaline phosphatase,

and urease in their respective medium by Bacillus sp., Pseudomonas sp., Azotobacter

sp., and Actinomycetes the experiment was carried out at different agitation speeds

ranging from 120 to 180 rpm at 30 ºC, pH of 7.0 with 1% of inoculum for 48 hrs.

After 48 hrs, the supernatant samples were withdrawn after centrifugation at 8000

rpm for 20 min at 4 ºC and used as a crude enzyme solution for assaying different

enzymatic activities.

APPENDIX - V

1. GC-MS ANALYSIS REPRESENTING MAJOR CONSTITUENTS OF

METHANOLIC EXTRACTS OF GUT FLUIDS FROM E.FOETIDA

S.No.

Peak Name Molecular

Formula

Molecular

Weight

Retention

Time

Hit

Spectrum

%

Peak

Area

1. 1-Propenyl aziridine C5H9N 83 3.33 37016 1.83

2. 4-Octadecenal C18H34O 266 3.75 37039 1.86

3. Methanamine N-

methoxy C2H7NO 61 6.04 370151 2.24

4. Cyclohexasiloxane,

dodecamethyl C12H36O6Si6 444 7.20 370207 8.39

5.

2,3-Dihydro – 3, 5-

dihydroxy-6- methyl-

4H-pyran -4-one

C6H8O4 144 8.52 370271 4.79

6.

Tricyclo [8.4.1.1(3,8)]

hexadeca-

3,5,7,10,12,14- hexane-

2-one anti-

C10H19DO2 172 9.07 370300 1.03

7. Tetraadecamethyl cyclo

heptasiloxane C14H42O7 Si7 518 9.34 370312 1.49

8.

Butanamide 3-(1-oxo-2-

phenylethylhydrazono)-

N-(2-methylpropyl)-

C16H23N3O2 289 9.87 370337 3.30

9. Di-[1,3,2]-oxazino[6,5-f;

5’,6’-H] quinoxaline C36H46N6O2 594 10.40 370363 1.47

10. 2-Amino-3-Methyl-

Pentanoic Acid C6H13NO2 131 11.29 307409 5.37

11. DL-Isoleucine C6H13NO2 131 11.76 370432 4.18

12.

1,2,4-Trioxolane-2-

octanoic acid, 5-octyl-,

methyl ester

C4H6C12O 344 12.64 370475 1.04

13. Rac-5-Oxopyrrolidine-2-

carbonsaure-methylester C6H9NO3 143 14.33 370557 0.96

14. Benzene Acetamide C8H9NO 135 14.70 370575 5.59

15. N-Hydroxymethyl-2-

phenylacetamide C9H11NO2 165 14.95 370577 1.96

16. Erucic acid C22H42O2 338 15.74 370626 2.18

17. Isorhoifolin C19H38O4 330 17.01 370688 1.18

18. Phenylalanine C9H11 NO2 165 18.50 370762 15.07

19.

1-Hexadecanaminium,

N,N,N,-trimethyl-,

bromide

C19H42N 284 20.47 370858 5.16

20. Cyclohexyl Carbamoyl

Azide C7H12N4O 168 23.47 3701005 3.41

21.

1,4-diaza-2,5,-dioxo-3-

isobutyl bicycle[4.3.0]

nonane

C11H18N2O2 210 24.97 3701078 4.36

22.

1,4,7- Triazaheptane,

1,7-BIS(1-Methyl-1-

Phosphonato) Ethyl-

C10H27N3O6P2 347 25.94 3701124 1.38

23.

1,4-diazo-2,5-dioxo-3-

isobutyp bicycle[4.3.0]

nonane

C11H18N2O2 210 27.06 3701179 8.06

24. 4,4’-Biphenol C12H10 154 27.77 3701214 1.46

25.

3,9-Diazatricyclo

[7.3.0.0(3,7)] Dodecan-

2,8-Dione

C10H14N2O2 194 28.33 3701242 3.42

26. Octadecane, 1-[2-

(hexadecycloxy)ethoxy]- C18H38O2 538 30.80 3701350 3.16

27.. Phenol, 4-methyl-2-[5-

(2-thienyl)pyrazol-3-yl]- C14H12N2OS 256 32.44 3701419 1.11

28.

1,4-diaza-2,5-dioxo-3-

isobutyl bicycle[4.3.0]

nonane

C11H18N2O2 210 32.96 3701440 1.30

29.

6-Ethoxy-3-Methyl-1,3-

Benzothiazol-2(3H)-

Imine

C10H12N2OS 208 35.09 3701527 1.48

30

3-benzyl-1,4-diaza-2,5-

dioxobicyclo[4.3.0]

nonane

C14H16N2O2 244 35.69 3701552 1.78

2. GC-MS ANALYSIS REPRESENTING MAJOR CONSTITUENTS OF

ETHANOLIC METHANOLIC EXTRACTS OF GUT FLUIDS FROM

E.FOETIDA

S.No.

Peak Name

Molecular

Formula

Molecular

Weight

Retention

Time

Hit

Spectrum

% Peak

Area

1. 1-Propenylaziridine C5H9N 83 3.33 37116 1.18

2. Cyclopentasiloxane, decamethyl C10H30O5 Si5 370 4.92 37193 1.52

3. Desulphosinigrin C10H17NO6S 279 6.10 371154 0.77

4. Cyclohexasiloxane,

Dedecamethyl- C12H36O6 Si6 444 7.20 371207 7.72

5. 2,3-Dihydro-3,5-dihydroxy-

6mehyl-4H-Pyran-4-one C6H18O4 144 8.52 371271 3.61

6. Cycloheptasiloxane,

tetradecamethyl- C14H42O7 Si7 518 9.34 371312 1.39

7. Benzeneethanamine, N, a,a-

trimethyl C10H13NO3 195 9.85 371337 2.77

8. Di-[1,3,2]-oxazino[6,5-f; 5’,6’-

H] quinoxaline C6H13N O2 131 10.15 371351 3.13

9. 2-Amino-3-methyl-pentanoic

acid C6H13NO2 131 11.13 371398 7.55

10 DL-Norleucine C6H13NO2 131 11.58 371422 7.44

11. 1-Tetradecanol C14H30O 214 12.59 371652 0.63

12. Bacteriochlorophyll-c-stearyl C52H72Mg

N4O4 362 15.74 371626 1.99

13. 2-Morpholino ethane sulfonic

acid C27H32CIN2O4 252 16.29 371575 1.35

14. 1-Methyl-2-naphthylamine C11H11N 157 18.36 371753 1.28

15 Ethyl a-d-glucopyranoside C8H16O6 208 18.85 371778 16.04

16. Pyroquilon C11H11NO 173 20.29 371848 2.43

17.

Tertbutyloxy formamide N-

methyl-N-[4-(1-pyrloidinyl)-2-

butynyl)]-

C14H24N2O2 252 22.31 371923 1.17

18. Benzeneacetamide C8H9NO 135 14.70 371947 9.84

19. Dimethyl Ester of Eicosan-1,20-

Dioic Acid C18H34O4 314 23.10 371986 1.54

20. Cyclohexyl Carbomyl Azide C7H12N4O 168 23.47 3711005 1.34

21. Dodecyldiglycol C16H34O3 274 24.24 37111043 0.85

22. 1,4-diaza-2,5-dioxo-3-isobutyl

bicycle [4.3.0] nonane C11H18N2O2 210 24.98 3711078 2.65

23. N-Methyl-2-Propyl-5-

Butylperidine C13H27N 210 27.06 3711181 6.88

24. 3,9-Diazatricyclo[7.3.0.0(3,7)

Dodecan-2,8-Dione C10H14N2O2 194 28.36 3711244 2.49

25. Doconexent C22H32O2 344 30.05 3711320 3.49

26. Ethanol, 2-(octadecyloxy) C20H42O2 314 30.80 3711351 4.05

27. Perhydrocyclopenta[c]isoxazole,

4,5,6-tri(benzyloxy) C27H29NO4 342 31.47 3711377 0.86

28. Stigmast-5-en-3-ol, (3a)- C29H50O 414 32.33 3711414 2.10

29. 4-Formylfluorenone C14H8O2 208 35.10 3711526 0.68

30. 3-Benzyl-1,4-diaza-2,5-

dioxobicyclo [4.3.0) nonane C14H16N2O2 244 35.70 3711549 1.21

APPENDIX- VI

PHYSICAL PROPERTIES OF EXPERIMENTAL SOIL

SNo Properties Value

(%)

1. Coarse sand 47.77±0.122

2. Fine sand 36.45±0.128

3. Silt 5.65±0.107

4. Clay 9.26±0.112

5. Textural class Sandy

LA

ND

UT

ILIZ

AT

ION

PA

TT

ER

N I

N T

HE

ST

UD

Y A

RE

A (2

009-2

012)

S.N

o.

Pa

rtic

ula

rs

Yea

r 2

00

9-2

01

0

Yea

r 2

01

0-2

01

1

Yea

r 2

01

1-2

01

2

Ero

de

Dis

tric

t B

ha

va

ni

Ta

luk

E

rod

e D

istr

ict

Bh

av

an

i T

alu

k

Ero

de

Dis

tric

t

Bh

av

an

i T

alu

k

1.

Geo

gra

ph

ical

Are

a (h

a)

34

617

8.1

35

69

848

.21

0

34

617

8.1

35

69

848

.21

0

34

617

8.1

35

69

848

.21

0

2.

To

tal

No

. o

f V

illa

ges

3

75

5

7

37

5

57

3

75

5

7

3.

Net

Ag

ricu

ltu

ral

Are

a (h

a)

22

478

6.1

65

55

134

.93

5

22

159

5.1

80

54

343

.77

0

19

887

3.7

55

45

024

.93

0

4.

Fal

low

lan

d (

ha)

6

43

11

.44

2

55

60.7

77

66

764

.71

1

69

73.0

41

80

930

.51

9

12

898

.04

5

5.

To

tal

irri

gat

ed a

rea

(ha)

1

99

38

8.9

80

39

077

.44

5

19

724

7.7

25

46

096

.71

0

12

616

8.3

65

40

583

.46

5

6.

Are

a u

nd

er f

ore

sts

(ha)

1

42

5.0

00

Nil

1

42

5.0

00

Nil

1

42

5.0

0

Nil

7.

Cu

ltiv

able

la

nd

(h

a)

19

93

88

.98

0

47

13

9.2

20

1

97

247

.72

5

46

09

6.7

10

1

83

29

9.5

65

4

05

83

.46

5

8.

To

tal

Rai

nfa

ll (

mm

) 7

10

.6

Nil

1

03

7.8

N

il

76

2.6

N

il

9.

To

tal

Man

ure

sto

res

53

4

68

54

1

68

54

1

69

Cro

ps

(i)

Pad

dy

(h

a)

38

113

.27

0

56

81.1

60

36

658

.05

5

59

28.3

70

33

719

.38

0

54

50.8

55

(ii)

. T

urm

eric

(h

a)

45

765

.30

5

51

75.9

30

39

241

.66

7

47

86.4

48

31

338

.20

3

37

50.1

10

(iii

).

Rag

i (h

a)

67

26

.95

0

18

61

.72

0

64

41

.45

5

16

14

.335

5

45

8.8

60

1

55

0.2

60

(iv

) M

aize

(h

a)

13

781

.00

1

57

12.0

10

12

574

.50

5

50

18.9

35

12

584

.51

0

37

68.3

25

(v)

Ben

gal

gra

m (

ha)

0

.57

0

Nil

N

il

Nil

N

il

Nil

(vi)

G

rou

nd

nu

t (h

a)

19

392

.44

0

46

43.8

50

19

894

.58

5

54

23.5

05

18

534

.59

0

41

86.0

50

(vii

) S

ug

arca

ne

(ha)

3

45

96

.57

0

10

905

.92

0

34

478

.79

5

10

274

.41

0

31

544

.77

5

95

34.6

95

(vii

i)

Veg

etab

les

(ha)

1

22

48

.41

5

28

78.8

10

1

23

30

.55

5

32

80

.82

5

89

10

.05

5

21

40

.95

5

(ix

) O

il S

eed

s (h

a)

44

258

.99

0

12

114

.02

5

43

219

.88

5

12

029

.16

0

38

852

.54

2

77

08.6

50

(x)

Oth

er n

on

-foo

d c

rop

s (h

a)

19

02.1

20

37

8.6

40

16

51.4

85

34

9.9

65

18

62.5

50

36

2.1

85

(xii

) C

ott

on

(h

a)

14

21.0

05

84

9.2

55

13

46.8

05

76

7.5

90

84

5.7

25

36

4.8

25

So

urc

e: D

istr

ict

Sta

tist

ica

l O

ffic

e, E

rod

e D

istr

ict.

APPENDIX – VII

ANALYTICAL METHODS FOR PHYSICO-CHEMICAL PARAMETERS

1. Determination of pH

About 30 grams of the air dried vermicast sample was passed through a 2 mm

sieve to which 60 ml of distilled water was added in a 100 ml glass beaker. The

sample was stirred well and allowed to stand for half an hour. The electrode was

immersed into the beaker containing the sample suspension and meter readings were

recorded. The pH of the samples were determined by using a digital Elico pH meter

(Jackson, 1973).

2. Determination of electrical conductivity

Electrical conductivity is the measurement of the total amount of soluble salts

present in the sample and are expressed as millicimens / cm (mS/cm). To 5 g of the

vermicast sample, 50 ml of distilled water was added. The sample was stirred well

and allowed to stand for half an hour. The electrical conductivity of the sample was

determined by using a conductivity meter and the EC was read and expressed in

millicimens/cm (mS/cm) (Jackson, 1973).

3. Organic carbon

At each sampling period, the vermicompost sample was diluted with distilled

water in the ratio of 1:5 (w: v) and mixed well. From this, 0.5 ml of the supernatant

solution was incubated with 10 ml of 1N potassium dichromate and 20 ml of

concentrated sulphuric acid at room temperature for 30 min. The volume of the

mixture solution was made up to 200 ml with distilled water. To this, 10 ml of

orthophosphoric acid and a few drops of diphenylamine indicator were added. This

mixture was titrated against 0.5N ferrous ammonium sulphate solution. The end point

was the colour change from dark blue to green. From the titre value, the total organic

carbon content was calculated using the following formula (Walkley and Black,

1934).

Calculation

Organic carbon (%) = x

Where Y= Titrated value

X= Blank value

4. Estimation of total nitrogen

The nitrogen in the organic material are converted to ammonium sulphate by

conc. H2SO4 during digestion. This salt, on steam-distillation, liberates ammonia

which are collected in boric acid solution and titrated against the standard acid (Pellett

and Young, 1980).

Procedure

At each sampling period, 100 mg of the vermicompost samples were taken

and digested in 30 ml digestion flasks with 2.0 g of potassium sulphate, 80.0 mg of

mercuric oxide, 2 ml of conc. H2SO4 and boiling chips till the solution turned

colourless. After cooling the digest, it was diluted with a small quantity of distilled

ammonia – free water and transferred to the distillation apparatus.

The kjeldahl flask was rinsed with successive small quantities of water and

placed in 100 ml conical flasks containing 5 ml of boric acid solution with a few

drops of mixed indicator with the tip of the condenser dipping below the surface of

the solution. Ten ml of 40% sodium hydroxide thiosulphate solution was added to the

test solution in the apparatus. It was distilled and the ammonia was collected in 10 ml

of 2 % boric acid. The tip of the condenser was rinsed and the solution was titrated

against the standard acid which until the first appearance of violet colour, which was

the end point. A reagent blank was run with an equal volume of distilled water and

subtracted the titration volume from that of the sample titre volume.

Calculation

The nitrogen content in the samples was calculated based on the following

formula

Total Nitrogen/Kg=

5. C:N ratio

The C:N ratio was calculated by dividing organic carbon and total nitrogen

content of the sample

6. Estimation of total phosphorus

The heteropoly compound produced by the reaction between

vanadomolybdate and phosphate radical in nitric acid medium are yellow in colour.

The intensity of the yellow colour are read using a colorimeter at 470nm (Jackson,

1973).

6.1.Procedure

6.1. (a). Preparation of standard

0.2 g of pure KH2PO4 was dissolved in 400ml of distilled water. To this, 25

ml of 7N H2SO4 was added and the total volume was made up to 1000 ml to get 50

ppm of phosphorus (50µg/ml). Phosphorus standards ranging from 0 to 20 ppm were

prepared. From this, 5 ml of the solution was pipetted into flask and the volume was

made to 50 ml with distilled water to result in, 50 ppm of phosphorus. Five ml of this

solution was transferred to a 25 ml volumetric flask, and 2.5 ml of Barton’s reagent

was added and the volume made upto 25 ml. The intensity of the colour for of each

standard was measured using a colorimeter at 470 nm wavelength and a standard

graph was drawn.

6.1. (b). Testing of samples

From the prepared vermicomposts sample, 1.0 ml was added to 15 ml of the

triple acid mixture in a 100 ml conical flask. The mixture was digested over a heated

sand bath, and the volume made up to 500 ml with distilled water. From this, 25 ml of

the mixture was pipetted out into volumetric flask to which, 2.5 ml of the Bartons

reagent was added. The volume of the mixture was made upto 250 ml with distilled

water. After few minutes, the intensity of yellow colour developed was read at 470

nm in a colorimeter. From the standard graph, the concentration of phosphorus in the

sample was read in ppm.

Calculation

Weight of the sample taken = ‘W’ g (1g)

Volume of the triple acid extracts =’V’ ml (50)

Aliquot taken for the colour development = 5ml

Corresponding from the standard graph = ppm

P = x x x 100

P = x x x 100

7. Estimation of total sodium and total potassium

In flame photometry, the test solution is carefully passed under controlled

conditions as a very fine spray in the air supply to a burner. In the flame, the solution

evaporates and the salt dissociates to given natural atoms. A very small proportion of

this salt gets into a higher energy state. When these excited atoms fall back to the

ground state, the light emitted is of a characteristic wavelength which are measured

(Jackson, 1973).

Procedure

At each sampling period, the vermicompost sample was diluted with distilled

water in the ratio of 1:5 (w: v) and mixed well. From this, 1ml of freshly prepared

supernatant of the vermicompost sample was taken in the microkjeldahl flasks to

which was added 12 ml of triple acid (9:2:1 ratio of concentrated nitric acid:

concentrated sulphuric acid: perchloric acid). The sample was digested over a heated

sand bath and made upto 100ml with distilled water.

For potassium estimation, 1 ml of the freshly prepared supernatant from the

vermicomposts sample was mixed with 100 ml of triacid mixture (concentrated nitric

acid, 60% perchloric acid and concentrated sulphuric acid in the ratio of 10:4:1) in

100 ml flask and the volume was made upto 50 ml with distilled water. The sodium

and potassium contents were fed directly to the flame photometer after adjusting it to

zero with blank and standardizing it with 100 ppm of sodium and potassium solution

with 100 as the galvanometer reading. The readings were noted. From the standard

graph drawn, the corresponding ppm was read and the percentage of sodium and

potassium were calculated.

Calculation

Na content (%) = ppm x 100 x 100

Potassium content (%) = ppm x

Weight of the samples taken = ‘W’ (g)

Volume made upto = ‘V’ (ml)

Content of K or Na in sample material

With reference to standard graph = x x 100

The percentage of K or Na = x

8. Estimation of total calcium

The pH of the sample are sufficiently increased (12 – 13) in order to

precipitate the magnesium as hydroxide, and only calcium is allowed to react with the

EDTA in the presence of a selective indicator (Jackson, 1973).

Procedure

During each sampling period, the vermicompost sample was diluted with

distilled water in the ratio of 1:5 (w: v) and mixed well. From this, 5.0 ml of freshly

prepared supernatant of the vermicompost sample was added to the porcelain basin

along with 50ml of the triple acid extracts and 10ml of NaOH followed by drop by

drop to neutralize the acidity (red litmus turns blue). The pH of the mixture was

adjusted to 12. A pinch of mureoxide indicator was later added to the mixture and

titrated against 0.02 N EDTA till the red colour changed from pinkish red to violet. A

blank without the sample was titrated and the volume of 0.02 N EDTA consumed was

noted.

Calculation

The percentage of calcium content was calculated by using the formula

Ca% = B x x x x 100

B- Volume of 0.02 M EDTA used for calcium

9. Estimation of iron, manganese, zinc and copper

The technique involves determination of concentration of a substance by the

measurement of absorption of the characteristic radiation resulting from the atomic

vapour of an element. When radiation that is characteristic to a particular element

passes though the vapour of the same element, absorption of radiation occurs in

proportion to the concentration of the atoms in the light path. The source of

characteristic radiation is a hollow cathode lamp, the cathode being made of the

element desired to be estimated (Lindsey and Norwell, 1978).

Procedure

At each sampling period, the vermicompost sample was diluted with distilled

water in the ratio of 1:5 (w: v) and mixed well. From this, 1.0 ml of the sample was

added to a microkjeldahl flask along with 12 ml of triple acid and digested over a

heated sand bath. The volume was made up to 100 ml with distilled water. The

components as required were directly fed to the atomic absorption spectrophotometer

at wave length of 248.3, 213.9, 279.5 and 324.8, corresponding to iron, manganese,

zinc and copper respectively. The available concentrations (ppm) of iron, manganese,

zinc and copper were calculated from the standard graph.

10. Estimation of total carbohydrate

Carbohydrates are first hydrolyzed into simple sugars using dilute

hydrochloric acid. In a hot acidic medium glucose is dehydrated to hydroxymethyl

furfural. This compound reacts with anthrone and forms a green coloured product

with an absorption maximum at 630 nm (Hedge and Hofreiter, 1962)

Procedure

After each sampling period, the vermicompost sample was diluted with

distilled water in the ratio of 1:5 (w: v) and mixed well. From this, 1 ml of the sample

was taken in boiling tubes and hydrolyzed with 5.0 ml of 2.5 N HCl by keeping them

in a boiling bath for 3 hours and cooled to room temperature. Then, it was neutralized

with solid sodium carbonate until the effervescence ceased. The mixture was made

upto 100 ml with distilled water and centrifuged. The supernatant was collected and

0.5 ml and 1.0 ml of aliquots were pipetted out into test tubes for analysis. The

standards were prepared by taking 0, 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the working

standard. ‘0’ served as the blank and the volume of the solution was made up to 1.0

ml in all the tubes including the sample tubes by adding distilled water.

To this, 4.0 ml of the anthrone reagent was added and the tubes were heated

for 8 min in a boiling water bath. The tubes were cooled rapidly and the green to dark

green colour formed were read at 630 nm. The standard graph was drawn by plotting

the concentration of the standard on X-axis versus absorbance on the Y-axis. From the

graph the amount of carbohydrate present in the sample tubes were calculated.

Calculation

Amount of carbohydrate present in 1.0 g of the

sample = x 100

5. Estimation of cellulose

Cellulose is the most abundant organic compound in nature. It is a major

structural polysaccharide in plant cell walls, made up of D-glucose units which are

linked to each other by -1, 4-glycosidic bond. Cellulose undergoes acetolysis with

acetic/nitric reagent to form acetylated cellodextrine which on dissolution is

hydrolyzed to form glucose units on treatment with 67% H2SO4. On dehydration with

H2SO4, glucose forms 5-hydroxymethyl furfural which on reaction with anthrone

gives a green colored product (Updegroff, 1969).

Procedure

After each sampling period, 1.0 g of the vermicompost sample was added

with 3 ml of acetic: nitric reagent ( mixed 150 ml of 80% acetic acid with 15 ml of

conc. HNO3) and mixed well using a vortexer. The mixture was placed in a water

bath at 100°C for 30 min after which it was cooled and centrifuged at 8000 rpm for

15-20 min. The supernatant was discarded and the residue was then washed with

water followed by the addition of 10 ml 67% H2SO4 and left for 1 hr. From this, 1ml

of this solution was taken and diluted with distilled water to 100 ml. From this

dilution to 1 ml of the sample was added 10 ml of the anthrone reagent (200 mg

anthrone dissolved in 100 ml of conc. H2SO4) and mixed well and heated in boiling

water bath for 10 min. The mixture was cooled and the absorbance measured at 630

nm against the blank containing anthrone reagent and water. The standard graph was

prepared using cellulose solution at varied concentrations (40-200 µg of cellulose).

6. Estimation of lignins

Lignins are phenolic polymers present in the cell walls of plants which are

responsible together with cellulose, for the stiffness and rigidity of plant stems.

Lignins are especially associated with woody plants, since upto 30% of the organic

matter of trees consists of lignin (Zedrazil and Brunnert, 1980).

Procedure

The residual lignocellulose from each sample was harvested by adding 50 ml

of distilled water, homogenized and filtered through pre-weighed filter paper. The

residual lignocellulose on filter paper was treated with 5 ml of concentrated H2SO4

(96.98%) and 25 ml of concentrated HCl (37%) for 16 hours at 25°C. The acidity of

the sample was adjusted to neutrality with Na2CO3 and the volume was made to 50 ml

with distilled water. The sample was then filtered through pre-weighted Whatman

No.1 filter paper and the residue was dried in hot air oven at 100°C over night and

weighed. The dried residue was ashed using a muffle furnace at 500°C for 5 hrs,

cooled in a desiccator and the weight of the ash was determined. Finally the actual

lignin content in the sample was measured by substracting the weight of the ash

content from the dried residual content.

APPENDIX – VIII

ANALYTICAL METHODS FOR VITAMINS

1. Estimation of vitamin A (Retinol)

The method is based on the measurement of the interaction of vitamin A with

trifluroacetic acid, the intensity of which is a function of the concentration of vitamin

A which are measured at 620 nm (Nield and Pearson, 1963)

Procedure

After each sampling period, the vermicompost sample was diluted with

distilled water in the ratio of 1:5 (w: v) and mixed well. From this, 1.0 ml was added

with 1.0 ml of the saponification mixture (2N/KOH in 90% alcohol) and heated under

gentle reflux for 20 min at 60ºC. The mixture was made up to 25 ml with distilled

water and kept in a separating funnel. The solution was then extracted thrice using 25,

15 and 10 ml of petroleum ether at 40 - 60ºC. The ether extracts were pooled and

washed in 50-100 ml of distilled water repeatedly until the wash water was free of

alkali. The petroleum ether extract was then dried by adding anhydrous sodium

sulphate. The volume of the extract was noted. From this, 3.0ml of petroleum ether

phase was pipetted out to a clean cuvette and read at 420 nm against petroleum ether

blank without delay to prevent evaporation of the solvent and destruction of

carotenoids by light. The reading was noted as A1. The -carotene working standards

were measured at 450nm.

The aliquots were evaporated to dryness at 60º C in a water bath. The residue

was taken immediately and 2.0 ml of TFA reagent was added to it. The mixtures were

rapidly transferred to the cuvette and the absorbance values measured at 620 nm and

the reading was noted as A2. The vitamin A working standard was read at 620 nm.

Calculation

For accurate calculation of the vitamin A content, it is necessary to correct the

absorbance by carotene at 620nm.

A3 = A2 – A1

Where,

A1 = Absorbance of carotene at 450 nm.

A2 = Absorbance at 620 nm due to both carotene and vitamin A

A3 = Absorbance at 620 nm of vitamin A

Amount of Retinol =

3 = Volume of petroleum ether from 1.0ml extract

2= Aliquot of the petroleum ether used for the assay

1 = 10% extract taken from initial sample

The results were expressed as ug/g.

2. Estimation of Vitamin E

Tocopherols can be estimated using Emmerie-Engel reaction, which are based

on the reduction of ferric to ferrous ions by tocopherols, which then form a red colour

with 2, 2’dipyridyl. Tocopherols and carotenes are first extracted with xylene and the

extraction read at 460 nm to measure carotenes. A correlation is made for these after

adding ferric chloride and the read at 520 nm (Varley, 1976).

Procedure

At each sampling period, the vermicompost sample was diluted with distilled

water in the ratio of 1:5 (w: v) and mixed well. From this, 1.5 ml of the sample was

made upto 3 ml with 1.5 ml of distilled water followed by the addition of 1.5 ml of

water, 1.5 ml of ethanol, and 1.5 ml of xylene to the sample. The mixture was

centrifuged at 10,000 rpm for 15 min. After centrifugation, 1.0 ml of the xylene layer

was transferred into another tube and 1.0 ml of 2, 2’ dipyridyl reagent was added and

mixed well. From this, 1.5 ml of the mixture was transferred into a colorimeter

cuvette, kept in room temperature for 15 minutes and the absorbance value of the test

sample and standard (10 mg/l) against the blank (0.33 ml of ferric chloride solution)

read at 520 nm.

Calculation

The amount of vitamin E was calculated using the formula,

Amount of tocopherol =

3. Estimation of Ascorbic acid (Vitamin C)

Ascorbic acid is first dehydrogenated by bromination. The dehydroascorbic

acid is then reacted with 2, 4 dinitrophenyl hydrazine to form osazone and dissolved

in sulphuric acid to give an orange-red colour solution which is measured at 540 nm

(Sadasivam and Manickam, 1992).

Preparation of sample

At each sampling period, the vermicompost sample was diluted with distilled

water in the ratio of 1:5 (w: v) and mixed well. From this, 10 ml of the sample was

transferred into 100 ml conical flask and bromine water was added drop by drop with

a constant mixing to remove enolic hydrogen atoms in ascorbic acid. (When the

extract turned orange yellow due to excess bromine, it was expelled by blowing in

air).Then, the mixture was made up to a known volume (25 or 50 ml) with 4%

ascorbic acid solution into dehydroform by bromination.

Procedure

Different aliquots (0.1 ml – 2 ml) of brominated sample extracts were taken in

test tubes. The volume in each tube was made up to 3 ml by adding distilled water and

one ml of the DNPH reagent followed by 1-2 drops of thiourea that was added to each

tube. A blank was prepared by using water in place of ascorbic acid solution. The

contents of the tubes were mixed well and incubated at 37ºC for 3 hours. The

resulting orange red osazone crystals were dissolved by adding 7 ml of 80% sulphuric

acid and the absorbance measured at 540 nm. The absorbance value was plotted on a

standard graph using various ascorbic acid concentrations (10-100 µg/ml) and the

ascorbic acid content in the sample was calculated.

4. Estimation of total phenol (Bray and Thorpe, 1954).

Phenol reacts with the oxidizing agent phosphomolybdate in the Folin-

Ciocalteau reagent under alkaline conditions and results in the formation of a blue

coloured complex, the molybdenum blue which is measured at 650 nm

calorimetrically.

Preparation of Sample

The extraction process was carried out using buffer for the enzyme assay.

About 100mg of the samples were taken and ground well with pestle and morter in

10ml of the buffer. The ground sample was then centrifuged and the supernatant

solution was collected for the protein estimation.

Procedure

From the extracted sample, 0.2 to 2 ml was taken in different test tubes and the

volume of each sample was made up to 3ml with distilled water. Then, to the sample

was added 0.5ml of the Folin-Ciocalteau reagent followed by the addition of 2 ml of

20% Na2CO3 solution to each tube and thoroughly mixed. The tubes were heated in a

water bath for exactly one minute. Then, the tubes were cooled and the absorbance

was measured at 650 nm against a reagent blank. The absorbance values were plotted

on a standard graph using different concentrations of catechol and the concentration

of total phenol in the test samples were noted and expressed as mg phenols/100mg

material.

5. Estimation of Humic acid content (HA)

The humic acid content in the sample was estimated by adopting the

procedure as described by Schnitzer, (1978).

Procedure

After each sampling period, the vermicompost sample was diluted with

distilled water in the ratio of 1:5 (w: v) and mixed well. From this, 5 ml was dissolved

in 100 ml of 0.5 N NaOH (Dissolved 20 gm of NaOH in 100 ml of dist.H2O). The

liquid was homogenized by vortexing and incubated at room temperature for 24 hrs.

After incubation, the liquid was filtered through Whatman No.1 filter paper. The

filtrate was collected in a jar, acidified with 6N HCl (Mixed 60ml of concentrated

HCl with 40 ml of dist.H2O) to pH 1. After 3 hrs, the coagulate was dialyzed against

dis. H2O till they were free from chloride and finally dried in a hot air oven at 40ºC.

The humic acid content in the test sample was expressed in mg/5ml substrates.

6. Estimation of Indole Acetic acid (IAA)

Indole acetic acid in the sample was determined in vitro by the method of

Brick et al. (1991). From the prepared sample, 5 ml was taken and centrifuged at

10,000 g for 10 min and the supernatant collected. Two ml of this supernatant was

allowed to react with 2 drops of orthophosphoric acid and 4 ml of Salkowski reagent

(1ml of 0.5 M FeCl3 in 50 ml of 35% HClO4) at 28±2ºC for 30 min. At the end of the

incubation, the development of a pink color indicated the presence of IAA.

Quantification of IAA was done by measuring the absorbance in a spectrophotometer

at 530 nm. Concentration of IAA in the sample was measured with the help of a

standard graph for IAA (Himedia) obtained in the range of 10-100 µg/ml.

7. Estimation of Gibberellic acid (GA3)

The method of Mali and Bodhankar (2009) with slight modifications was used

for the determination of gibberllins. From the prepared sample, 25 ml was taken into

25 ml of volumetric flask, mixed with 15 ml of phosphomolybdic acid reagent and

placed in a boiling water bath. After 1 hour, the temperature of the flask was reduced

to room temperature and then final volume was made to 25 ml with distilled water.

The absorbance of colour developed was measured at 780 nm in a spectrophotometer

using distilled water as blank and the concentration of gibberellins was calculated by

preparing standard curve by using gibberellic acid (GA3) as standard (10-100 µg/ml).

APPENDIX - IX

ANALYSIS OF PHYSICAL PARAMETERS OF FIELD SOIL

1. Determination of pH

About 30 grams of the air dried soil sample was passed through a 2mm sieve

which to 60 ml of distilled water was added in a 100 ml glass beaker. The sample was

stirred well and allowed to stand for half an hour. The electrode was immersed into

the beaker containing the sample suspension and meter readings were recorded. The

pH of the samples was determined by using a digital Elico pH meter (Jackson, 1973).

2. Determination of Electrical Conductivity

Electrical conductivity is the measurement of the total amount of soluble salts

present in the sample and are expressed as millicimens / cm (mS/cm). To 5g of the

soil sample, 50 ml of distilled water was added. The sample was stirred well and

allowed to stand for half an hour. The electrical conductivity of the sample was

determined by using a conductivity meter and the EC was read and expressed in

millicimens/cm (mS/cm) (Jackson, 1973).

3. Determination of Bulk Density

The bulk density of sample was analysed by using the core method. The

undisturbed core (5 cm diameter, 12 cm deep) soil samples were collected and bulked

for each plot. This was done at before and after the planting. Soil from core sampler

was transferred to a container and placed in an oven at 105°C, and dried until constant

weight. Dried soil weight was recorded and bulk density was calculated by the

formula of Black and Hartge (1986).

Bulk Density =

4. Determination of Porosity

The porosity of the test sample was observed by following the method of

Chandrabose et al. (1988). Twenty grams of the sample was weighed and transferred

in small quantities to a 100ml measuring cylinder. The volume of the sample was

noted. 50 ml of distilled water was added to the sample and soaked well. The cylinder

was then kept undisturbed for some time till the pore space was filled with water. The

volume of the sample and water were recorded at the end of the experiment and the

percentage of pore space was calculated accordingly;

Weight of the sample taken = W gm.

Volume of the sample = P ml.

Volume of water added = Q ml.

Volume of sample and water = P + Q ml.

Volume of sample and water at the end of the experiment = r ml.

Pore space (P + Q) - r = `t’ ml.

Percentage of pore space = t / p x 100

5. Determination of Moisture Content

The moisture content of the samples under study were determined by the

procedure of Chandrabose et al., (1988). The samples were collected in polythene

bags and closed rapidly and tightly. From this, 100 g of sample was taken in a

weighing bottle and placed in an electric oven at 105oC for about 24 hrs and the dry

weight of the sample was recorded. The loss in weight was calculated and expressed

in percentage using the following calculation:

Weight of the moisture bottle = A g

Weight of the moisture bottle + sample = B g

Weight of the sample taken = (A-B) g

Weight of moisture bottle and sample after drying in the oven = C g

Weight of moisture in the sample = (B-C) g

Percentage of moisture in the sample = x 100

6. Water Holding Capacity

The water holding capacity of the vermicompost samples under study were

determined by the method of Inbar et al. (1993). The sample was collected in

polythene bags and closed rapidly and tightly. From this, 100 g of the sample was

taken in a beaker and added with the required quantity of water till it became wet. The

percentage of water holding capacity of the samples were calculated using the

following formula

Water Holding Capacity (%) = (Wet weight – Dry weight) / volume of water x 100

APPENDIX - X

ESTIMATION OF TOTAL CHLOROPHYLL

Chlorophyll was extracted in 80% acetone and the absorption at 663nm and

645nm were read in a spectrophotometer. Using the absorption coefficients, the

amount of chlorophyll was calculated Arnon, (1949).

Procedure

One gram of the finely cut leaf samples after various treatments were taken

and ground well with pestle and morter with the addition of 20 ml of 80% acetone.

The ground samples were centrifuged at 5,000 rpm for 5 minutes and the

supernatant transferred into 100ml volumetric flasks. The residues were ground

again with 20ml of 80% acetone, centrifuged and the supernatants transferred to

the same volumetric flask. The same procedure was repeated until the residue was

colourless and the mortar and pestle were thoroughly washed with 80% acetone

and the collected clear washings transferred to the volumetric flask with the

volume being made up to 100 ml with 80% acetone. The absorbance value was

observed at 645, 663 and 652nm against the solvent (80% acetone) blank.

Calculation

The amount of total chlorophyll present in the extract (mg chlorophyll / g

tissue) was calculated using the following equations:

Total chlorophyll (mg) / g tissue = 20.2 (A645) - 8.02 (A663) x

Where, A= absorbance at specific wavelengths

V= final volume of chlorophyll extract in 80% acetone

W= fresh weight of the extracted tissue

APPENDIX XI

ECONOMICS OF VERMICOMPOST PRODUCTION

Vermicomposting Production Estimate per Annum

Production Capacity

Capacity : 600 Tonnes (2.5 tonnes a day)

Selling Price : Rs.1300 / Tonne

1. Project Cost/ Capital Investment

S.No.

Description

Amount (Rs)

Pre-Operative Expenses

1. Initial Capital 1,00,000.00

2. Working Capital for 1 month (25000 x 12) 3,00,000.00

Total Project Cost 4,00,000.00

2. Investment in Tools and Equipments

S.No.

Description

Contribution

Percentage

Amount (Rs)

1. Weighing machine

(Platform type)

1 1 8,500.00

2. Water pump & pipes for

water sprinkling

1 1 16,000.00

3. Tools & Equipments 1 set - 13,000.00

4. Hand driven chaff cutter 1 - 17,500.00

5. Vermicompost bed with

earthworms

10 (15

kg)

- 45,000.00

Total 1,00,000.00

3. Estimated Variable Expenses

S.No Description Unit Quantity Salary/

Month (Rs.)

Amount

(Rs.)

1. Sugarcane Trash Tonne 300 100.00 30,000.00

2. Cow dung Tonne 250 125.00 31250.00

3. Sugarcane Trash Tonne 300 100.00 30,000.00

4. Earthworms Kg 10 240.00 2,400.00

5. Power - - 12x 250.00 3,000.00

6. Water - - 12x 250.00 3,000.00

Postage & Stationary

Expenses

12x125.00 1,500.00

Transportation Expenses 12x1750 21,000.00

Advertisement Expenses 12x2100 25,200.00

Miscellaneous Expenses 12x350 4,200.00

Total 1,51,550.00

Expected Expenses for the production of 1 unit vermicompost =

= Rs. 252.58

3. Fixed Expenses

S.No Description Quantity Per Month Amount

(Rs)

1. Salary of supervisor 5,500.00 x12 66,000.00

2. Wage of skilled workers 3,000.00 x12 36,000.00

4. Rent of land & shed 3,500.00 x12 42,000.00

5. Depreciation

(a) Weighing machine (Platform type) @

10%

850.00

(b) Water pump & pipes for water

sprinkling @ 5%

1700.00

(c) Tools & Equipments @ 10% 1300.00

(d) Hand driven chaff cutter @ 10% 1750.00

(e) Vermicompost bed with earthworms

@ 10%

4500.00

6. Interest on Capital 4,00,000x10/100=

40,000

40,000.00

Total 1,94,100.00

4. Break even point table

Production/SalesFixed Cost

(Rs.)

Variable

Cost /Unit

(Rs.252.50)

Total Cost

(Rs.)

Sales

Rs.1300/unit

Loss/ BEP/

Profit

(Rs.)

0 1,94,100.00 - 1,94,100.00 - 1,94,100.00

(L)

50 1,94,100.00 12,625.00 2,06,725.00 65,000.00 1,41,725.00

(L)

100 1,94,100.00 25,250.00 2,19,350.00 1,30,000.00 89,350.00 (L)

150 1,94,100.00 37,875.00 2,31,975.00 1,95,000.00 36,975.00 (L)

185.31 1,94,100.00 46,807.00 2,40,907.00 2,40,907.00 BEP

200 1,94,100.00 50,500.00 2,44,600.00 2,60,000.00 15,400.00 (P)

250 1,94,100.00 63,630.00 2,57,730.00 3,25,000.00 67,270.00 (P)

300 1,94,100.00 75,750.00 2,69,350.00 3,90,000.00 1,20,650.00

(P)

350 1,94,100.00 88,375.00 2,82,475.00 4,55,000.00 1,72,525.00

(P)

400 1,94,100.00 1,01,000.00 2,95,100.00 5,20,000.00 2,24,900.00

(P)

450 1,94,100.00 1,13,625.00 3,07,725.00 5,85,000.00 2,77,275.00

(P)

500 1,94,100.00 1,26,250.00 3,20,350.00 6,50,000.00 3,29,650.00

(P)

550 1,94,100.00 1,38,875.00 3,32,975.00 7,15,000.00 3,82,025.00

(P)

600 1,94,100.00 1,51,200.00 3,45,300.00 7,80,000.00 4,34,700.00

(P)

Key: L: Loss; P: Profit

Marginal Cost Statement

Sales = Rs. 7,80,000.00

Loss Variable cost = Rs. 1,51,550.00

_______________

Contribution = Rs. 6,28,450.00

Loss Fixed Cost = Rs. 1,94,100.00

_______________

Profit = Rs.4,34,350.00

_______________

Profit Analysis

1. Profit Volume Ratio (P/V Ratio) =

=

2. Break Even Point

(a). In terms of Value =

= (or) = x 100

= Rs. 2,40,908.00

(b). In terms of quantity =

= = 185.31 Tonnes

3. Margin of Sales

(a). Actual Sales – BEP Sales = 7,80,000.00 – 2,40,908

= Rs. 5,39,09,092.00

(b). = x100 =Rs. 5, 39,096.00

APPENDIX X1I

MEDIA USED FOR THE MICROBIOLOGICAL EXAMINATIONS

Actinomycetes Isolation agar medium

Sodium casianate : 2.0g

L-Asparagine : 0.1g

Sodium propionate : 4.0g

Di-potassium phosphate : 0.5g

Magnesium phosphate : 0.1g

Ferrous sulphate : 0.001g

Agar : 15 g

Distilled water : 1000mL

pH : 7.0

Amylase enzyme production medium

Starch : 10g

Peptone : 10g

Yeast extract : 20.0g

K2HPO4 : 0.05g

MnCl2.4H2O : 0.015 g

MgSO4.7H2O : 0.25 g

CaCl2.2H2O : 0.05g

FeSO4.7H2O : 0.01g

Distilled water : 1000mL

pH : 7.1 0.2

Bismuth Sulphite agar medium

Peptic digest of animal tissue : 10g

Beef extract : 5.0g

Dextrose : 5.0g

Disodium phosphate : 4.0g

Ferrous sulfite : 0.3g

Bismuth sulfite agar : 8g

Brilliant green : 0.025g

Agar : 20g

Distilled water : 1000 mL

pH : 7.0±0.2

Cellulase enzyme production medium

Carboxy Methyl Cellulose : 10g

Peptone : 10g

K2HPO4 : 2.0 g

MgSO4. 7H2O : 0.3 g

(NH4)2SO4.7H2O : 2.5 g

Distilled water : 1000mL

pH : 7.1 0.2

Casaein agar medium

Peptone : 5.0g

Beef extract : 3.0g

Starch (soluble) : 2.0g

Agar : 15.0g

Distilled water : 1000 mL

pH : 7.0

Christensen medium

Urea : 20 g

Peptone : 1.0 g

KH2PO4 : 2.0 g

Glucose : 1.0 g

NaCl : 5.0 g

Agar : 15 g

Distilled water : 1000 mL

Phenol red indicator : 0.012 g

pH : 6.8±0.2

Christensen’s agar medium

Pancreatic Digest of Gelatin : 1.0g

Dextrose : 1.0g

Sodium chloride : 5.0g

Potassium phosphate : 2.0g

Urea : 20.0g

Phenol red : 0.012g

Agar : 15.0g

Distilled water : 1000mL

pH : 6.8±0.2

Crawford agar medium

Na2HPO4 : 1.5g

KH2PO4 : 4.0g

MgSO4.7H2O : 2.0g

NaCl : 0.2g

Agar : 20g

Saw dust : 0.1%

Distilled water : 1000 mL

pH : 6.8±0.2

EMB agar medium (Hi media)

Peptone : 10.0g

Lactose : 10g

K2HPO4 : 2.0g

Eosin Y : 0.4g

Methylene Blue : 0.065g

Agar : 15.0 g

Distilled water : 1000ml

pH : 7.1 0.2

Glucose Asparagines agar medium

Glucose : 10g

Asparagine : 0.5g

K2HPO4 : 0.5 g

Agar : 15 g

Distilled water : 1000 mL

pH : 7.0 ±0.2

MR-VP medium

Peptone : 0.5g

Dextrose : 5.0g

Potassium phosphate : 5.0g

Distilled water : 1000 mL

pH : 7.1±2

Mannitol Ashby agar medium

Mannitol : 20g

Potassium phosphate : 0.20g

Magnesium sulfate : 0.20g

Sodium chloride : 0.20g

Potassium sulfate : 0.10g

Calcium carbonate : 5.0g

Agar : 15.0 g

Distilled water : 1000mL

pH : 7.0 0.2

Malate Medium

L Malic acid : 5g

Potassium hydrogen

orthophosphate : 0.5g

Magnesium sulphate : 0.2 g

Sodium chloride : 0.1 g

Calcium chloride : 0.02g

Trace elements solution : 2 ml

Vitamin solution : 2 ml

Bromo thymol blue (0.5% aqueous

solution in 0.2 N KOH) : 2 ml

Potassium hydroxide : 4.98 g

Agar : 20 g

Distilled water : 1000ml

pH : 6.8 with KOH

Trace elements Solution

Sodium molybdate : 200 g

Manganous sulphate : 235 mg

Boric acid : 8 mg

Zinc sulphate : 24 mg

Distilled water : 200 ml

Vitamin Solution

Biotin : 10 mg

Pyridoxin : 20 mg

Distilled water : 100 ml

Nutrient agar medium

Peptone : 5.0 g

Beef extract : 3.0 g

Yeast extract : 3.0 g

Sodium chloride : 5.0 g

Agar : 20 g

Distilled water : 1000 mL

pH : 7.0±0.2

Pikovskaya’s agar medium

Yeast extract : 0.50g

Dextrose : 10.00g

Calcium phosphate : 5.00gm

Ammonium sulfate : 0.50g

Potassium chloride : 0.20g

Magnesium sulphate : 0.10g

Manganese sulfate : 0.0001g

Ferrous sulphate : 0.0001g

Agar : 15.00g

pH : 7.1 0.2

Protease enzyme production medium

Glucose : 1.0g

Peptone : 10.0g

K2HPO4 : 0.5g

MgSO4 : 0.1g

Distilled water : 1000mL

pH : 7.1 0.2

Sabouraud’s Dextrose Agar (g/L-1

)

Peptone : 10g

Glucose : 40g

Agar : 15 g

Distilled water : 1000 mL

pH : 5.6±0.2

Salkowsky indicator

Conc. HCl : 150 mL

FeCl2 (0.5M) : 7.5 mL

Distilled water : 250 mL

Salmonella-Shigella agar medium

Peptic digest animal tissue : 5.0g

Protease peptone : 5.0g

Beef extract : 5.0g

Lactose : 10g

Bile salt mixture : 8.5g

Ferric citrate : 10g

Sodium thiosulphate : 8.5g

Ferric citrate : 1.0 g

Brilliant green : 0.00033g

Neutral red : 0.025g

Agar : 15g

Distilled water : 1000 mL

pH : 7.0 0.2

Simmons’ Citrate agar medium

Ammonium di-hydrogen phosphate: 1.0g

Di-potassium phosphate : 1.0

Sodium chloride : 5.0g

Sodium citrate : 2.0g

Magnesium sulfate : 0.2g

Agar : 15.0g

Bromothymol blue : 0.08g

Distilled water : 1000mL

pH : 7 0.3

SIM agar

Peptone : 30.0g

Beef extract : 3.0g

Ferrous ammonium sulfate : 0.2g

Sodium thiosulfate : 0.025g

Agar : 3.0g

pH : 7.1 0.2

Starch agar Medium

Peptone : 5.0 g

Beef extract : 3.0 g

Soluble Starch : 2.0g

Agar : 15.0 g

pH : 7.0

Tributyrin agar medium

Lipase enzyme production medium

Peptone : 5g

Yeast extract : 3 g

Sodium chloride : 5 g

Vegetable oil : 10 ml

Distilled water : 1000mL

pH : 7.1 0.2

Tryptone broth

Tryptone : 10.0g

Calcium chloride (reagent) : 0.01-0.03

Sodium chloride : 5.0g

Distilled water : 1000mL

pH : 7.1 0.2

Urease enzyme production medium

D-glucose : 20.0 g

Peptone : 10.0g

Yeast extract : 5 g

KH2PO4 : 2.0 g

NaCl : 5 g

NaAc : 2 g

Urea : 20.0 g

MnSO4 : 0.05 g

NiSO4 : 0.05 g

Distilled water : 1000 mL

pH : 5.5

Xylan agar medium

Xylanase enzyme production medium

Xylan : 10 g

Yeast extract : 2 g

Magnesium sulphate : 0.2 g

Di-potassium hydrogen phosphate: 0.1 g

Potassium di-hydrogen phosphate: 0.5 g

Calcium chloride : 0.1 g

Distilled water : 1000mL

pH : 7.1 0.2

YEM broth

K2HPO4 : 0.04g

KH2PO4 : 0.01g

MgSO4.7H2O : 0.02g

NaCl2 : 0.01g

CaCl2 : 0.01 g

Maleic acid : 0.25 g

Yeast extract : 0.05g

pH : 7 0.3

Yeast Extract Mannitol agar medium

Peptone : 5.0g

Beef extract : 3.0g

Sodium chloride : 5.0g

Yeast extract : 5.0g

Calcium carbonate : 1g

Magnesium sulfate : 0.2g

Mannitol : 10g

Distilled water : 1000 mL

Agar : 15g

Congored (1% solution) : 2.5 mL

pH : 6.8 0.3