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