Chapter IV - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/6718/9/09_chapter 4.pdf · isolates had shown positive reaction with the appearance of blue-black colour ... Earthworm

111

Chapter – IV

RESULTS

112

4.1 Nitrogen mineralization potentials of Rhizospheric, Non-Rhizospheric soils

and Vermicompost:

4.1a. Chemical characterization of Rhizospheric, Non-Rhizospheric soils and

Vermicompost

The percent macronutrient analysis indicated that vermicompost consisted of

1.30 % N; 1.0 % P; 1.5 % K; 6.2 % Ca and 3.4 % Mg. These values are higher than

rhizospheric and non- rhizospheric soils. On the other hand non – rhizospheric soil

consisted of 54.2 % organic matter and 36.6% organic carbon. These values are

higher than that of rhizospheric soil and vermicompost respectively. Further

vermicompost showed a narrow range of C:N ratio (22:1) and C:P ratio (33:2) to

rhizospheric and non-rhizospheric soils (Table 1).

Table 1: Chemical characterization of Rhizospheric, Non-Rhizospheric soils and

Vermicompost

Parameter Non Rhizospheric soil Rhizospheric soil Vermicompost

pH 6.4 6.4 7.4

Organic matter (%) 54.2 24 37.8

Organic Carbon (%) 36.6 10.2 31.4

TKN (%) 1.02 0.48 1.30

Total Nitrogen (%) 0.075 0.043 0.102

NH4+ - N (µg) 223 110 164

NO3- - N (µg) 125 95 170

C:N ratio 31:1 37:1 22:1

C:P ratio 39:1 48:1 33:2

P (%) 0.68 0.80 1.0

K (%) 0.43 0.60 1.5

Ca (%) 3.3 4.3 6.2

Mg (%) 1.9 2.5 3.4

Fe (ppm) 1100 1200 1700

Mn (ppm) 373 380 475

Cu (ppm) 74 79 110

Zn (ppm) 341 345 450

B (ppm) 62 69 84

Values are the mean of three observations.

113

4.2. Collection of Earthworm gut homogenate:

Earthworms (Eisenia foetida) were collected from vermicompost pits of

S.V.Agricultural University, Tirupati and subjected to starvation for 24 hrs to exclude

the microbes associated with soil and other organic waste of the gut. After starvation

the earthworms were dissected out and used for isolation of indigenous gut

microflora.

4.2.1 Isolation of bacteria from earthworm gut homogenate by dilution plate

method:

Serially diluted gut homogenate was plated on Nutrient Agar (NA) and

incubated at 370C for 24 hrs for the isolation of bacteria. A total of ten distinctly

variable colonies based on their morphology were sub cultured and preserved for

further studies.

4.2.2. Primary screening of the isolates for the ability of nitrification:

All the ten bacterial isolates were subjected to primary screening and tested for

the production of nitrites and nitrates.

4.2.2a. Determination of Nitrite production:

After 3 weeks of incubation, the inoculated broth was tested for the presence

of nitrite using Trommsdorf‟s reagent and sulfuric acid. Among the ten isolates six

isolates had shown positive reaction with the appearance of blue-black colour

indicating the presence of nitrite in the medium.

114

4.2.2b. Determination of Nitrate production:

After 3 weeks of incubation, the inoculated broth was tested for the presence

of nitrate using Diphenylamine reagent and sulfuric acid. Among the ten isolates five

isolates had shown positive reaction with the appearance of deep blue colour

indicating the presence of nitrate in the medium.

Out of the ten isolates, four isolates were in common showing a positive

reaction for the both nitrite and nitrate and selected for the secondary screening.

4.2.3 Secondary screening of the isolates for the ability of nitrification:

The four isolates (1- 4) selected through the primary screening were subjected

to secondary screening to determine the best isolate based on its ability to nitrification

through microtiter plate method. Ammonium – calcium carbonate medium was placed

into each of the 8 by 12 wells of a sterile microplate. Aliquots of the four test isolates

of the earthworm gut (0.05ml) were pipetted into each of the first eight wells. Serial

dilutions were then performed by using sterile micropipettes calibrated to deliver

0.05ml and were rotated rapidly. Then the dilutions were further moved to the next

eight wells where they were again rapidly rotated. This process was continued until

serial dilutions have been carried out across the plate. The result obtained was 12 two

fold serial dilutions, with eight replicates at each dilution.

After inoculation and the performance of serial dilutions, the plates were

covered with polypropylene tape and incubated for 3 weeks. Three replicates were

maintained in order to note the weekly reports, because conflicting reports existed

concerning the optimum incubation time for ammonia and nitrite oxidizing

microorganisms [Curtis, et al., (1975), Matulewich, et al., (1975) ].

115

At the end of the incubation period each plate was scored by adding an

indicator (0.2gm of diphenyl amine in 100ml of concentrated H2SO4) to test for the

presence of nitrate and/or nitrite at room temperature (Morgan, 1930).

A blue color reaction indicated that these end products had been formed and

the well was scored as positive and all the four isolates had shown a positive result.

The absence of a blue color was scored as negative. The MPN values were calculated

according to the table provided by de Man (1975) and Parnow (1972) where the table

represents MPN values and standard errors, for a two fold dilution series with eight

tubes per dilution.

The codes P1, P2, P3 represents the number of positive wells in three

successive dilutions, where P1 corresponds to the highest dilution at which all wells

gave positive readings or to the dilution showing the highest number of positive wells

and results were mentioned in table 2 (Fig 3).

Table 2: Ability of nitrification by the four (1, 2, 3, 4) isolates of Earthworm gut:

Sample

Incubation

time

(in weeks)

No.of positive wells in

each dilution

MPN value

MPN

value in

the

original

inoculum

*P1

(32)**

*P2

(64)**

*P3

(128)**

Isolate 1 After 2 weeks 8 7 7 2.921 3738.88




*Dilution code **Dilution factor

116

Earthworm gut isolate 1 was positive within 2 weeks of incubation period as

indicated by the results shown in P1, P2, P3 dilutions the number of positive wells

according to the MPN table were 8, 7, 7 respectively and the value obtained was

2.921.

The MPN value was then multiplied by the dilution factor for P2, in this case

dilution factor of P2 is 64 hence a value of 186.944 was obtained. To calculate the

MPN in 1 ml of the original inoculum, this number is multiplied by 20 (i.e., 20 x

186.944 = 3738.880). Hence, the MPN for 1 ml of the original inoculum (isolate 1)

was 3738.880.

4.3. Identification of bacterial isolate:

Morphological, Cultural and Biochemical characterization of the selected

bacterial isolate was carried out according to the guidelines of Bergey‟s Manual of

Systemic Bacteriology (Volume II) and Manual of Medical Microbiology (Mackie

MacCartney, 1989).

Morphological studies had revealed that the isolate 1 was aerobic endospore

forming, non pigmented and wrinkled colony with concentric rings. The organism

was positive for growth also, under anaerobic conditions. The growing cells were

Gram positive, motile with rod shape. It is positive for Catalase, Methyl red, Voges

proskauer, Citrate utilization, Urease, Nitrate reduction, H2S production, Casein

hydrolysis, Starch hydrolysis; Degradation of Tyrosine, Lecithinase, Gelatin

liquefaction, Arginine dihydrolysis, Lipase, Chitinase and Phosphate solubilisation

reactions. The isolate 1 was also positive for the utilization of sugars like, glucose,

glycerol, maltose and starch. Negative towards oxidase, indole, utilization of

arabinose, xylose, lactose and mannitol (Fig. 4). The isolate grew well in nutrient

117

broth at pH range of 5.7 to 8.0 and showed salt tolerance at NaCl concentration upto

8% (w/v). Bacterial growth was observed in the temperature ranging from 100C -

450C with an optimum growth around 37

0C.

Table 3: Morphological and biochemical tests for identification of bacterial

isolate:

Identification tests Results of the Bacterial isolate

Colony morphology

Configuration

Margins

Surface

Pigmentation

Turbidity

Opacity

Gram‟s reaction

Cell shape

Size(μm)

Spores

Motility

Physiological tests

Growth at temperatures

50C

100C

300C

370C

400C

450C

500C

Growth in NaCl (%) concentration

2

5

7

Wrinkled, cream, round , concentric

Smooth

Butyraceous

-

+

Translucent

Positive

Rods

3-5μm in length, 1.0 -1.2 μm in width

+

+

-

+

+

+

+

+/W

-

+

118

10

Growth at pH

4

5

6

7

8

Growth under anaerobic condition

Biochemical tests

Indole test

Methyl red test

Voges proskauer test

Citrate utilization test

H2S production

Gelatin hydrolysis

Urea hydrolysis

Starch hydrolysis

Lectinase

Lipase (Tween 80 hydrolysis)

Catalase test

Oxidase test

Denitrification

Arginine dihydrolase

Phosphate solubilization

Chitinase

Casein hydrolysis

Degradation of Tyrosine

Nutritional characteristics

Starch

Maltose

Glucose

Glycerol

+

+

-

W

+

+

+

+

+

-

+

+

+

+

+

+

+

+

+

+

-

+

+

+

+

+

+

+

+

119

Succinate

β-alanine

L-histidine

L-lucine

D-alanine

Antibiotic resistance

Penicillin G

Ampicillin

Chloramphenicol

Erythromycin

Streptomycin

Tetracycline

Gentamycin

Tobramycin

Rifampicin

Polymyxin

+

+

-

-

-

-

-

-

+

+

+

-

-

+

+

+

+

4.4. Molecular Characterization of the isolate:

Based on the morphological, biochemical and physiological tests performed

for the isolate 1, it was identified as Bacillus sps., which was further confirmed at

species level by the molecular characterization.

4.4.1. PCR amplification of 16S rRNA

PCR amplification of 16S rRNA was carried out for the isolate 1 using

universal primers as described in the methods. A product of 495 bp was obtained from

the isolate. The optimum temperature for amplification of the gene was 55°C and

maximum product was 30 μg/ml. PCR product was purified and sequenced by using

DNA sequencer as already described. The BLAST search analysis of the 16S rRNA

120

gene sequence of the isolate was done against the Bacillus sps. The BLAST results

revealed 99.77 % homologous with Bacillus cereus.

Multiple sequence alignment of the isolate with closely related homologous

species was done with CLUSTAL W. Phylogenetic tree (Figure 1) was constructed

using Neighbour Joining Method. The 16S rRNA sequence of the isolate was

genotypically similar to Bacillus cereus.

Table 4: BLAST search results of the 16S rRNA sequence of Bacillus cereus

Fig 5 : Phylogenetic tree of isolate 1 ( Using Neighbour joining method).

4.5 Physiological conditions, Carbon and Nitrogen sources and C/N ratio

influencing the growth of the isolate:

Influence of physiological conditions like Temperature, pH, NaCl conc.,

Carbon and Nitrogen sources and C/N ratio were studied for the optimum growth of

the organism. A temperature of 370C, pH 7, NaCl 2%, Glucose as carbon source,

Peptone as nitrogen source and C/N ratio of 0.5% of Glucose: 1 % of peptone were

found optimum for the growth of B. cereus. The growth was measured by taking O.D

values at 600 nm for turbidity.

4.5.1. Effect of temperature on growth

Incubation temperature had influenced the metabolic reactions through

enzymatic activities which effected the growth of organism. B. cereus had produced

maximum growth when incubated at 370C and weak growth at 50

0C (Fig 6)

121

Figure 6: Effect of temperature on growth of B. cereus

0

0.5

1

1.5

2

2.5

3

25 30 35 37 40 45 50

Temperature (°C)

OD

at

600 n

m

4.5.2. Effect of pH on growth

The pH of the medium played an important role in the growth and metabolism

of the organism. B. cereus produced maximum growth at pH 7 and least growth at pH

10 (Fig 7)

Fig 7: Effect of pH on growth of B. cereus

0

0.2

0.4

0.6

0.8

1

1.2

1.4

4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

pH

OD

at

600 n

m

122

4.5.3. Effect of NaCl concentration on growth

Growth of the B. cereus was studied in Nutrient broth with 2 to 10% NaCl

concentration (Fig 8). NaCl concentration of 2% was found to be the optimum for

growth and decreased growth was observed from 4 % NaCl concentration.

Fig 8: Effect of NaCl concentration on growth of B. cereus

0

0.5

1

1.5

2

2.5

Control 2 4 6 8

NaCl Concentration (%)

OD

at

600 n

m

4.5.4. Effect of different carbon sources on growth of B. cereus

Eight different carbon sources, like – glucose, starch, sucrose, maltose,

lactose, raffinose, D-mannitol and arabinose were amended in NB medium to find out

suitable carbon source for optimum growth of B. cereus (Fig 9). Glucose was found to

be the best carbon source for the maximum growth of B. cereus isolate followed by

Starch, Maltose, Sucrose and Raffinose when compared to the rest of the carbon

sources.

123

Fig 9: Effect of carbon sources on growth of B. cereus

0

0.4

0.8

1.2

1.6

Gluco

se

Sta

rch

Suc

rose

Malto

se

Lactos

e

Raffi

nose

D- M

annito

l

Ara

binos

e

Cont

rol

Carbon sources

OD

at

600 n

m

4.5.5. Effect of nitrogen sources on growth of B. cereus

Provision of utilizable nitrogen source to organisms was the basic requirement

for the optimum growth; hence the NB medium was supplemented with different

nitrogen sources namely L-aspargine, L-alanine, L-arginine, L-proline, KNO3,

CaNO3, peptone and yeast extract. Among the nitrogen sources tested in the present

study, Peptone had shown high influence on the growth of B. cereus followed by

CaNO3 and Yeast extract when compared with the rest of nitrogen sources (Fig 10).

Fig 10: Effect of nitrogen sources on growth of B. cereus

0

0.4

0.8

1.2

1.6

L-Asp

argine

L-A

lanine

L-Histid

ine

L-Pro

line

L-Arg

inine

Soy

a bea

n m

eal

KNO3

CaN

O3

Pep

tone

Yea

st e

xtra

ct

Nitrogen sources

OD

at

600 n

m

124

4.5.6 Effect of different C/N ratios on growth

Different carbon and nitrogen sources were amended to find out suitable C/N

ratio for the optimum growth of B. cereus. Among the different ratios 0.5% of glucose

along with 1% of peptone had shown as the best ratio for the growth of B. cereus

(Fig 11).

Fig 11: Effect of different C/N ratio on growth of B. cereus

0

0.5

1

1.5

2

2.5

3

0.5:0.5 0.5:1 01:01 01:00.5 control

C:N ratio on growth

OD

at

600 n

m

4.6.1.1. Selection of suitable medium for ammonium oxidation by nitrifying B.

cereus

Nutrient availability was one of the major factors that influence the metabolic

activity of bacteria. Number of selective media have been developed and used for

studying the metabolic activities of nitrifying bacteria. In the present study five

different media like: the Modified Winogradsky‟s medium; medium recommended by

IMTECH; Stephenson‟s medium; Lewis and Pramer‟s medium and ACC medium,

were used for comparison of the growth and either ammonia oxidation by B.cereus

with incubation time.

125

Of the five media used for the metabolic activity of ammonium oxidizers the

ACC medium was found to be the best choice (Table 5.1). The nitrite released from

the added ammonium in different media was in the order ACC > Modified

Winogradsky‟s medium > IMTECH medium > Stephenson‟s medium > Lewis and

Pramer‟s medium (Fig 12).

Fig 12: Comparison of Different media for Ammonium oxidation

0

1000

2000

3000

4000

5000

6000

1 2 3 4 5 6

INCUBATION TIME (in Days)

Mic

rog

ram

s o

f N

O2-

- N

/ 50m

l

ACC Modified Winodradsky's medium

IMTECH Stephenson's medium

Lewis and Pramer's medium

Table 5: Results of Two Way ANOVA

Source of variation Sum of

Squares df Mean Square F-value p-value

Media 39366043.600 4 9841510.900 17.102*

* 0.000

Incubation Time 23594145.656 5 4718829.131 8.200** 0.000

Error 46036053.733 80 575450.672

Total 132772855.000 90

** Significant at 1% level

126

Table 5.1: Average Concentration of Nitrite in different media

Media Mean production of Nitrite

Lewis and Pramer's medium 51.78a

Stephenson's medium 56.89a

IMTECH 200.61a

Modified Winogradsky's medium 457.11a

ACC 1803.56b

Note: Same letter indicates insignificant difference in any pair of Media according to

Duncan's Multiple Range Test (DMRT).

The results obtained for the selection of suitable medium for ammonia

oxidizer were tabulated. (Table 5). The results were showing a significant difference

at 1% level among the media since P value 0.000 < 0.01 for the corresponding F-

value (17.102).

Further, Post hoc test i.e., Duncan‟s Multiple Range Test (DMRT) was carried

out to observe the pair wise difference of media with respect to the incubation time

and were found significant. The result suggested that the difference between any two

media with respect to the incubation time was significant which means the ammonium

oxidation levels were varying according to the medium (Table 5.1).

4.6.1.2. Selection of suitable medium for nitrite oxidation by B. cereus

In order to identify an enriched medium which would support high metabolic

activity of nitrite oxidizers, three media i.e., NCC, Modified Winogradsky‟s medium

and IMTECH medium were used for the metabolic activity of nitrite oxidizer. The

NCC medium was found to be the best choice (Table 6). The nitrate release from the

added nitrite in different media with time were in the order NCC > Modified

Winogrdsky‟s medium > IMTECH medium (Fig 13).

127

The decrease in the nitrite content from the initially available amounts in the

medium was taken as a measure for the increase in the metabolic activity of the

nitrifying bacteria (Fig 13). There was no trace of nitrite in Modified Winogradsky‟s

medium after 14 days of incubation and where as about 2% of initially added nitrite

remained after 14 days of incubation in the IMTECH medium. The results showed

that NCC medium was the best choice for nitrite oxidation since nitrite was oxidized

by 9th

day.

Fig 13: Comparison of different media for nitrite oxidation

Comparision of Different media for Nitrite Oxidizers

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Incubation period (in Days)

% o

f N

itri

te N

itro

gen

NCC Modified Winodradsky's medium IMTECH

Table 6: Results of Two Way ANOVA

Media Mean Std.

Deviation F-value

p-

value

NCC 100 0

374851.75** 0 Modified Winodradsky's medium 74.96 0.057

IMTECH 52.9 0.1


The results obtained for the selection of suitable medium for nitrite oxidation

were tabulated (Table 6). The results were showing a significant difference at 1%

128

level among the media since P value 0.000 < 0.01 for the corresponding F- Value

(374851.75).

4.6.2. Nitrogen mineralization by B. cereus :

Nitrogen mineralization was studied by estimating the initial ammonium

levels to the oxidised nitrite and nitrate levels.

4.6.2.1. Estimation of nitrite

Nitrite production by B. cereus increased with incubation time upto 9 days and

later there was a gradual decrease in the nitrite production (Fig 14) .

Fig 14: Nitrite production by B. cereus

0

20

40

60

80

100

2 3 4 5 6 7 8 9 10 11 12

Sampling period (In Days)

Co

ncen

trati

on

of

Nit

rite

(µ

g/g

)

4.6.2.2. Estimation of nitrate

Nitrate production by B. cereus gradually increased with incubation period

upto 12th

day after which there was a gradual decrease in nitrate production (Fig 15).

129

Fig 15: Nitrate production by B. cereus

0

40

80

120

160

200

2 3 4 5 6 7 8 9 10 11 12 13 14

Sampling period (In Days)

Co

ncen

trati

on

of

Nit

rate

(µ

g/g

)

4.7. Factors effecting nitrification activity:

The influence of different physiological factors like carbon source, nitrogen

source, temperature, pH, metal ions, inhibitors, chealating agents and pesticides on

nitrification was observed.

4.7.1a. Effect of carbon sources on nitrite production:

Five carbon sources (1% w/v) like glucose, sodium acetate, L-lysine, malate

and citrate were amended in 250 ml of Basal mineral salts medium to find out

suitable carbon source for the production of nitrite. Sodium acetate was found to be

the best carbon source for the maximum production of nitrite followed by citrate and

glucose when compared to the other carbon sources (Fig 16).

130

Fig 16: Effect of Carbon sources on Nitrite production

0

10

20

30

40

50

60

70

80

90

100

2 3 4 5 6 7 8 9


Co

ncen

trati

on

of

Nit

rite

(µ

g/g

)

Glucose Sodium acetate L-lysine Malate Citrate

Table 7 : Results of Two Way ANOVA


Squares Df

Mean

Square F-value p-value

Carbon sources 5520.450 4 1380.113 91.289*

* 0.000

Incubation Time 38656.500 7 5522.357 365.282

** 0.000

Error 1632.750 108 15.118

Total 346410.00

0 120

** Significant at 1% level.

Table 7.1 : Average concentration of Nitrite with different carbon sources

Carbon source Mean production of Nitrite

L-lysine 40.50a

Malate 46.00b

Glucose 48.88c

Citrate 55.00d

Sodium acetate 59.88e



The results obtained for the selection of suitable carbon source for the

production of nitrite was tabulated (Table 7). The results were showing a significant

131

difference at 1% level among the carbon sources since P value 0.000 < 0.01 for the

corresponding F- value (365.282).


out to observe the pair wise difference of carbon sources with respect to the

incubation time and were found significant. The result suggested that the difference

between any two carbon sources with respect to the incubation time was significant

which means the nitrite production levels were varying according to the carbon source

(Table 7.1).

4.7.1b. Effect of carbon sources on nitrate production:

Five carbon sources (1% w/v) like glucose, sodium acetate, L-lysine, malate

and citrate were amended in 250 ml of Basal mineral salts medium to find out

suitable carbon source for the production of nitrate. Sodium acetate was found to be

the best carbon source for the maximum production of nitrate followed by citrate and

glucose when compared to the other carbon sources (Fig 17).

Fig 17: Effect of Carbon sources on Nitrate production

0

20

40

60

80

100

120

140

160

180

200

2 3 4 5 6 7 8 9 10 11 12 13 14


Co

ncen

trati

on

of

Nit

rate

(µ

g/g

)

Glucose Sodium acetate L-lysine Malate Citrate

132


Source of

variation Sum of Squares Df

Mean


Carbon

sources 159356.031 4 39839.008 316.160** 0.000

Incubation

Time 251124.738 12 20927.062 166.076** 0.000

Error 22429.569 178 126.009

Total 2425743.000 195

** Significant level at 1% level

Table 8.1: Average concentration of Nitrate with different carbon sources

Carbon Sources Mean production of Nitrate

L-lysine 59.08a

Malate 74.85b

Glucose 118.23c

Citrate 122.69c

Sodium acetate 130.62d



The results obtained for the selection of suitable carbon source for the

production of nitrate was tabulated (Table 8). The results were showing a significant

difference at 1% level among the carbon sources since P value 0.000 < 0.01 for the

corresponding F- Value (316.160).


out to observe the pair wise difference of carbon sources with respect to the


between any two carbon sources with respect to the incubation time was significant

which means the nitrate production levels were varying according to the carbon

source (Table 8.1).

133

4.7.2a. Effect of nitrogen sources on production of Nitrite:

The effect of various nitrogen sources (1% w/v) such as Tryptone, Beef

extract, Ammonium sulphate, Urea, Soya bean meal and Peptone were amended in

250 ml of Basal mineral salts medium to find out the suitable nitrogen source for the

production of nitrite was determined. Among the nitrogen sources tested in the

present study, Ammonium sulphate had shown high influence on the production of

nitrite followed by Urea, Tryptone and others (Fig 18).

Fig 18: Effect of Nitrogen sources on Nitrite production

0

10

20

30

40

50

60

70

80

90

100

2 3 4 5 6 7 8 9


Co

ncen

trati

on

of

Nit

rite

(µ

g/g

)

Tryptone Beef extract Ammonium sulphate Urea Soya bean meal Peptone Control


:

Source of


Mean


Nitrogen

sources 52548.321 6 8758.054 165.519** 0.000

Incubation

Time 37490.089 7 5355.727 101.218** 0.000

Error 8148.536 154 52.913

Total 380349.000 168

**Significant at 1% level

134

Table 9.1: Average concentration of Nitrite with different Nitrogen sources

Nitrogen sources Mean production of Nitrite

Control 0.00a

Beef extract 40.50b

Soya bean meal 42.38b

Peptone 46.00c

Tryptone 46.25c

Urea 53.50d

Ammonium sulphate 58.25e



The results obtained for the selection of suitable nitrogen sources for the


difference at 1% level among the nitrogen sources since P value 0.000 < 0.01 for the



out to observe the pair wise difference of nitrogen sources with respect to the


between any two nitrogen sources with respect to the incubation time was significant

which means the nitrite production levels were varying according to the nitrogen

source (Table 9.1).

4.7.2b. Effect of different Nitrogen sources on production of Nitrate:

The effect of various nitrogen sources (1% w/v) such as Tryptone, Beef

extract, Ammonium sulphate, Ammonium chloride, Urea, Soya bean meal, Peptone,

Sodium nitrite, KN02, and CaN02, were amended in 250 ml of Basal mineral salts

medium to find out the suitable nitrogen source for the production of nitrate. Among

135

the nitrogen sources tested in the present study, Sodium nitrite had shown highest

influence on the production of nitrate followed by Calcium nitrite and Potassium

nitrite and other nitrogen sources (Fig 19).

Fig 19: Effect of Nitrogen sources on Nitrate production

0

20

40

60

80

100

120

140

160

180

200

2 3 4 5 6 7 8 9 10 11 12 13 14


Co

ncen

trati

on

of

Nit

rate

(Mic

rog

ram

s/G

ram

)

Tryptone Beef extract Ammonium sulphate Ammonium chloride

Urea Soya bean meal Peptone Potassium Nitrite

Sodium Nitrite Calcium Nitrite Control


Source of


Mean


Nitrogen

sources 579494.811 10 57949.481 293.020** 0.000

Incubation

Time 280978.993 12 23414.916 118.397** 0.000

Error 80293.007 406 197.766

Total 3180282.000 429


136

Table 10.1: Average concentration of Nitrate with different Nitrogen sources

Nitrogen sources Mean production of Nitrate

Control 0.00a

Beef extract 50.00b

Soya bean meal 54.46c

Peptone 56.23c

Tryptone 56.69c

Ammonium chloride 58.77c

Urea 69.69d

Ammonium sulphate 76.69e

Potassium Nitrite 119.31f

Calcium Nitrite 122.69f

Sodium Nitrite 130.23g



The results obtained for the selection of suitable nitrogen source for the

production of nitrate was tabulated (Table 10). The results were showing a significant

difference at 1% level among the nitrogen sources since P value was 0.000 < 0.01 for

the corresponding F- value (293.020).


out to observe the pair wise difference of nitrogen sources with respect to the


between any two nitrogen sources with respect to the incubation time was significant

which means the nitrate production levels were varying according to the nitrogen

source (Table 10.1).

137

4.7.3. Effect of pH on nitrification activity

The effect of pH on nitrification activity was determined by using (NH4)2SO4

and NaNO2 as substrates in the Basal mineral salts medium.

4.7.3a. Effect of pH on Nitrite production

The pH of the medium played an important role in the production of nitrite in

the medium. Maximum levels of nitrite was produced by B. cereus at pH 7 followed

by 8, 9 and least at pH 12 (Fig 20).

Fig 20: Effect of pH on Nitrite production

0

10

20

30

40

50

60

70

80

90

100

2 3 4 5 6 7 8 9


Co

ncen

trati

on

of

Nit

rite

(µ

g/g

)

6 5 8 7 9 10 11 12 Control



Squares Df Mean Square F-value p-value

pH 8138.250 7 1162.607 90.943** 0.000

Incubation Time 53910.000 7 7701.429 602.432** 0.000

Error 2262.750 177 12.784

Total 468378.000 192


138

Table 11.1: Average concentration of Nitrite at different pH

pH Mean production of Nitrite

12 38.50a

11 40.38a

5 40.50a

10 42.38b

6 46.25c

9 47.25c

8 53.50d

7 58.25e

Note: Same letter indicates insignificant difference in any pair of Media for pH

according to Duncan's Multiple Range Test (DMRT).

The results obtained for the selection of suitable pH for the production of

nitrite was tabulated (Table 11). The results were showing a significant difference at

1% level among the different pH since P value 0.000 < 0.01 for the corresponding F-

value (90.943).


out to observe the pair wise difference of pH with respect to the incubation time and

were found significant. The result suggested that the difference between any two pH

ranges with respect to the incubation time was significant which means the nitrite

production levels were varying according to the pH range (Table 11.1).

4.7.3b. Effect of pH on Nitrate production

The pH of the medium played an important role in the production of nitrate

and B. cereus produced maximum levels of nitrate at pH 7 followed by 8, 6 and least

at pH 5. (Fig 21).

139

Fig 21: Effect of pH on Nitrate production

0

20

40

60

80

100

120

140

160

180

200

2 3 4 5 6 7 8 9 10 11 12 13 14


Co

ncen

trati

on

of

Nit

rate

(µ

g/g

)

5 6 8 7 9 10 11 12 Control


Source of variation Sum of Squares Df Mean Square F-value p-value

pH 321661.356 7 45951.622 284.542** 0.000

Incubation Time 269407.212 12 22450.601 139.019** 0.000

Error 47156.019 292 161.493

Total 2830131.000 312


Table 12.1: Average concentration of Nitrate at different pH

pH Mean production of

Nitrate

5 50.00a

12 56.23b

11 57.54b

10 58.54b

9 75.92c

6 118.77d

8 122.31d

7 131.23e

Note: Same letter indicates insignificant difference in any pair of Media for pH

according to Duncan's Multiple Range Test (DMRT).

140

The results obtained for the selection of suitable pH for the production of

nitrate were tabulated (Table 12). The results were showing a significant difference at

1% level among the pH since P value 0.000 < 0.01 for the corresponding F- value

(284.542).


out to observe the pair wise difference of pH with respect to the incubation time and

were found significant. The result suggested that the difference between any two pH

ranges with respect to the incubation time was significant which means the nitrate

production levels were varying according to the pH range (Table 12.1).

4.7.4. Effect of temperature on nitrification activity

The effect of Temperature on nitrification activity was determined by using

(NH4)2SO4 and NaNO2 as substrates in the Basal mineral salts medium.

4.7.4a. Effect of Temperature on Nitrite production

The production of nitrite at different temperatures played an important role

in the medium. B. cereus produced maximum levels of nitrite at 370C, followed by

400C and least at 60

0C (Fig 22).

Fig 22: Effect of Temperature on Nitrite production

0

10

20

30

40

50

60

70

80

90

100

2 3 4 5 6 7 8 9


Co

ncen

trati

on

of

Nit

rite

(µ

g/g

)

30 35 37 40 45 50 55 60 Control

141




Temperature 8138.250 7 1162.607 90.943** 0.000

Incubation Time 53910.000 7 7701.429 602.432** 0.000

Error 2262.750 177 12.784

Total 468378.000 192


Table 13.1: Average concentration of Nitrite at different temperatures

Temperature (0C) Mean production of Nitrite

60 38.50a

55 40.38a

35 40.50a

30 46.25b

45 47.25b

40 53.50c

37 58.25c



The results obtained for the selection of optimum temperature for the


difference at 1% level among the temperatures since P value 0.000 < 0.01 for the



out to observe the pair wise difference of temperatures with respect to the incubation

time and were found significant. The result suggested that the difference between any

two temperature ranges with respect to the incubation time was significant

which means the nitrite production levels were varying according to the temperature

(Table 13.1).

142

4.7.4b. Effect of Temperature on Nitrate production

The production of nitrate at different temperatures played an important role in

the medium. B. cereus produced maximum levels of nitrate at 370C followed by 40

0C,

350C and least at 60

0C (Fig 23).

Fig 23: Effect of Temperature on Nitrate production

0

20

40

60

80

100

120

140

160

180

200

2 3 4 5 6 7 8 9 10 11 12 13 14

Incubation perid (in Days)

Co

ncen

trati

on

of

Nit

rate

(µ

g/g

)

30 35 37 40 45 50 55 60 Control




Temperature 316916.740 7 45273.820 289.583** 0.000

Incubation Time 269338.212 12 22444.851 143.563** 0.000

Error 45651.635 292 156.341

Total 2823813.000 312


143

Table 14.1: Average concentration of Nitrate at different temperatures

Temperature (0C) Mean production of Nitrate

60 50.00a

55 57.23b

30 57.31b

50 58.69b

45 75.69c

35 119.23c

40 122.31c

37 130.08c



The results obtained for the selection of optimum temperature for the

production of nitrate were tabulated (Table 14). The results were showing a

significant difference at 1% level among the temperature since P value 0.000 < 0.01

for the corresponding F- value (289.583).


out to observe the pair wise difference of temperatures with respect to the incubation


two temperatures with respect to the incubation time was significant which means the

nitrate production levels were varying according to the temperature (Table 14.1).

4.7.5. Effect of metal ions on nitrification activity

The effect of different metal ions on nitrification activity was determined by

using (NH4)2SO4 and NaNO2 as substrates in the Basal mineral salts medium.

144

4.7.5a. Effect of metal ions on Nitrite production

Supplementation of culture medium with metal cations improved substantially

the production of nitrite. This observation strongly suggested the requirement of some

metal ions for production of nitrite. The presence of Mg+2

, Ca+2

followed by Fe+3

and

Co+2

at 5.0 mM enhanced the production of nitrite compared with rest of the metal

ions (Fig 24).

Fig 24: Effect of Metal ions on Nitrite production

0

10

20

30

40

50

60

70

80

90

100

2 3 4 5 6 7 8 9

Incubation (in Days)

Co

ncen

trati

on

Nit

rite

(µ

g/g

)

Ca2+ Mg2+ Hg2+ Co+2 Cd+2 Fe+3 Cu2+ Zn2+ Control




Metal ions 10811.583 8 1351.448 111.794** 0.000

Incubation Time 60675.625 7 8667.946 717.026** 0.000

Error 2417.750 200 12.089

Total 535665.000 216


145

Table 15.1: Average concentration of Nitrite production in presence of different

Metal ions

Metal ions Mean production of

Nitrite

Zn2+

37.00a

Cd2+

39.75b

Cu2+

41.13b

Hg2+

43.00c

Control 44.25c

Co2+

46.25d

Fe3+

49.50e

Ca2+

55.13f

Mg2+

60.13g



The results obtained for the effect of metal ions on the production of nitrite

was tabulated (Table 15). The results were showing a significant difference at 1%

level among the metal ions since P value 0.000 < 0.01 for the corresponding F- value

(111.794).


out to observe the pair wise difference among the metal ions with respect to the


between any two metal ions with respect to the incubation time was significant which

means the nitrite production levels were varying according to the metal ion (Table

15.1).

146

4.7.5b. Effect of metal ions on Nitrate production

Supplementation of culture medium with metal cations improved substantially

the production of nitrate. This observation strongly suggested the requirement of some

metal ions for production of nitrate. The presence of Mg+2

, Ca+2

and Fe+3

at 5.0 mM

enhanced the production of nitrate compared with other metal ions (Fig 25).

Fig 25: Effect of Metal ions on Nitrate production

0

20

40

60

80

100

120

140

160

180

200

2 3 4 5 6 7 8 9 10 11 12 13 14

Incubation (in Days)

Co

ncen

trati

on

of

Nit

rate

(µ

g/g

)

Ca2+ Mg2+ Hg2+ Co+2 Cd+2 Fe+3

Cu2+ Zn2+ Control




Metal ions 318995.270 8 39874.409 282.458** 0.000

Incubation Time 328451.816 12 27370.985 193.887** 0.000

Error 46444.769 329 141.170

Total 3095736.000 350


147

Table 16.1: Average concentration of Nitrate production in presence of different

Metal ions

Metal ions Mean production of Nitrate

Zn2+

50.31a

Cd2+

57.31b

Cu2+

57.46b

Hg2+

58.54b

Control 74.23c

Co2+

75.71c

Fe3+

119.38d

Ca2+

122.69d

Mg2+

129.69e



The results obtained for the effect of metal ions on the production of nitrate


level among the metal ions since P value 0.000 < 0.01 for the corresponding F- value

(282.458).


out to observe the pair wise difference of metal ions with respect to the incubation


two metal ions with respect to the incubation time was significant which means the

nitrate production levels were varying according to the metal ion (Table 16.1).

148

4.7.6. Effect of Inhibitors and Chealating agents on nitrification activity

The effect of different Inhibitors and Chealating agents on nitrification activity

was determined by using (NH4)2SO4 and NaNO2 as substrates in the Basal mineral

salts medium.

4.7.6a. Effect of Inhibitors and Chealating agents on Nitrite production

The effect of various Inhibitors/ Chealating agents (5mM) such as Nitrapyrin,

Dicyanodiamide, Thiourea, Potassium cyanide, Sodium diethyl dithiocarbonate and

L- Histidine in the basal mineral salts medium on nitrite production was determined.

Out of all, Nitrapyrin had shown the profound effect on Nitrite production (Fig 26).

Fig 26: Effect of Inhibitors/Chealators on Nitrite production

0

10

20

30

40

50

60

70

80

90

2 3 4 5 6 7 8 9

Incubation time (in Days)

Co

ncen

trati

on

of

Nit

rite

(µ

g/g

)

Nitrapyrin Dicyanodiamide Thiourea

Potassium cyanide Sodium diethyldithiocarbonate L-Histidine

Control


Source of variation Sum of Squares Df Mean


Inhibitors/Chealators 50369.786 6 8394.964 109.668** 0.000

Incubation Time 474.375 7 67.768 0.885 0.520

Error 11788.500 154 76.549

Total 94809.000 168


149


Inhibitors/Chealators

Inhibitors/Chealators Mean production of

Nitrite

Nitrapyrin 4.00a

Sodium diethyldithiocarbonate 5.38b

Thiourea 6.13b

Dicyanodiamide 6.50b

Potassium cyanide 8.88b

L-Histidine 10.00b

Control 56.00c



The results obtained for the effect of Inhibitors/Chealators on the production

of nitrite was tabulated (Table 17). The results were showing a significant difference

at 1% level among the Inhibitors/Chealators since P value 0.000 < 0.01 for the



out to observe the pair wise difference of Inhibitors/Chealators with respect to the


between any two Inhibitors/Chealators with respect to the incubation time was

significant which means the nitrite production levels were varying according to the

Inhibitors/Chealators (Table 17.1).

150

4.7.6b. Effect of different Inhibitors and Chealating agents on Nitrate production

The effect of various Inhibitors/ Chealating agents at 5mM such as Nitrapyrin,

Dicyanodiamide, Thiourea, Potassium cyanide, Sodium diethyl dithiocarbonate and

L- Histidine in the Basal mineral salts medium was determined. Out of all Nitrapyrin

had shown the profound effect on Nitrate production (Fig 27).

Fig 27: Effect of Inhibitors/Chealators on Nitrate production

0

20

40

60

80

100

120

140

160

180

2 3 4 5 6 7 8 9


Co

ncen

trati

on

of

Nit

rate

(µ

g/g

)

Nitrapyrin Dicyanodiamide Thiourea

Potassium cyanide Sodium diethyldithiocarbonate L-Histidine

Control



Squares df

Mean


Inhibitros/Chealators 184815.321 6 30802.554 106.384** 0.000

Incubation Time 1976.089 7 282.298 0.975 0.452

Error 44589.536 154 289.542

Total 353691.000 168


151


Inhibitors/Chealators

Inhibitros/Chealators Mean production of

Nitrate

Nitrapyrin 10.25a

Sodium diethyldithiocarbonate 11.25a

Thiourea 11.50a

Dicyanodiamide 14.75a

Potassium cyanide 15.38a

L-Histidine 17.75a

Control 108.00b



The results obtained for the effect of Inhibitors/ Chealators on the production

of nitrate were tabulated (Table 18). The results were showing a significant difference

at 1% level among the Inhibitors/ Chealators since P value 0.000 < 0.01 for the



out to observe the pair wise difference of Inhibitors/ Chealators with respect to the


between any two Inhibitors/ Chealators with respect to the incubation time was

significant which means the nitrate production levels were varying according to the

Inhibitors/ Chealators (Table 18.1).

152

4.7.7 Effect of pesticidies on nitrification activity

In order to determine the effect of pesticides on nitrification, Endosulfan, a

chlorinated organic insecticide; Dithane M-45 (Mancozeb), a contact fungicide and

Neem oil (Azadirachtin), a biological insecticide, were selected in the present study.

Stock solutions of pesticides were prepared by dissolving in distilled water.

4.7.7a. Effect of pesticides on nitrite production

ACC medium of 50 ml was dispensed in each of the 100 ml conical flasks and

sterilized. After cooling, 2 ml of 24 hrs culture was inoculated into the medium. The

known concentrations of pesticides 10 µg/ml were added to the flasks. The flask

containing medium and inoculum, but without a pesticide served as control. All the

flasks were incubated at 370C in an orbital shaker at 120 rpm. Nitrite levels were

estimated daily for a period 12 days (Fig 28).

Fig 28: Effect of pesticides on Nitrite production

0

10

20

30

40

50

60

70

80

90

100

2 3 4 5 6 7 8 9 10 11 12

Incubationperiod (in Days)

Co

ncen

trati

on

of

Nit

rite

(Mic

rog

ram

s/G

ram

)

Control (without Pesticide) Endosufan Mancozeb Neem oil

153



Squares df

Mean


Pesticide 49271.455 3 16423.818 207.007** 0.000

Incubation Time 10515.409 10 1051.541 13.254** 0.000

Error 9362.045 118 79.339

Total 188670.000 132



pesticides

Pesticide Mean production of Nitrite

Endosulfan 16.18a

Mancozeb 18.82a

Neem oil 22.00b

Control (without Pesticide) 63.36c



The results obtained for the effect of pesticides on the production of nitrite

was tabulated (Table 19). The results were showing significant difference at 1% level

among the pesticides since P value 0.000 < 0.01 for the corresponding F- value

(207.007).


out to observe the pair wise difference of pesticides with respect to the incubation


two pesticides with respect to the incubation time was significant which means the

nitrite production levels were varying according to the pesticides (Table 19.1).

154

4.7.7b. Effect of pesticides on nitrate production

NCC medium of 50 ml was dispensed in each of the 100 ml conical flask and

sterilized. After cooling, 2 ml of 24 hrs culture was inoculated into the medium.

The known concentrations of pesticides at 10, 25, 50 µg/ml were added to the flasks.

The flask containing medium and inoculum, but without a pesticide served as control.

All the flasks were incubated at 370C in an orbital shaker at 120 rpm. Nitrate levels

were estimated daily for a period 14 days (Fig 29).

Fig 29: Effect of Pesticides on Nitrate production

0

20

40

60

80

100

120

140

160

180

200

2 3 4 5 6 7 8 9 10 11 12 13 14


Co

ncen

trati

on

of

Nit

rate

(Mic

rog

ram

s/G

ram

)

Control (without Pesticide) Neem oil Endosufan Mancozeb



Squares Df

Mean


Pesticide 118032.818 3 39344.273 285.381** 0.000

Incubation Time 110650.909 10 11065.091 80.260** 0.000

Error 16268.182 118 137.866

Total 1019286.000 132


155


pesticides

Pesticide Mean production of Nitrate

Endosulfan 48.55a

Mancozeb 55.27b

Neem oil 77.64c

Control

(without Pesticide) 124.91d



The results obtained for the effect of pesticides on the production of nitrate


level among the pesticides since P value 0.000 < 0.01 for the corresponding F- value

(285.381).


out to observe the pair wise difference of pesticides with respect to the incubation


two pesticides with respect to the incubation time was significant which means the

nitrate production levels were varying according to the pesticides (Table 20.1).

4.8. Purification and Characterization of Nitrifying Enzymes:

4.8.1. Partial Purification and characterization of Ammonium Monooxygenase

enzyme:

4.8.1a. Purification:

The cell free culture filtrate obtained by sonication and was suspended in 10

mM Tris HCl was used as crude enzyme source. In the culture filtrate, total protein

content was determined by the methods described in section 3.9.1b. The supernatant

was fractionated by precipitation with ammonium sulphate at 80% saturation. The

156

precipitate was suspended in 20 mM Tris HCl buffer (pH 8.0) and dialyzed against

the same buffer. After dialysis, protein content was determined. Ammonium sulphate

fraction (40 ml) had total protein of 140 mg and specific activity of 3.9 EU/mg.

Fraction from ammonium sulphate precipitation was subjected to Ion-

exchange chromatography by means of a DEAE- Sepharose CL6B. Total activity and

protein content for the 14 ml fraction obtained from DEAE- Sepharose CL6B column

chromatography were showing a total activity of 400 EU and 29 mg of total protein

respectively. The specific activity calculated was 13.8 EU/mg for DEAE- Sepharose

CL6B fraction.

The fraction from DEAE- Sepharose CL6B chromatography was subjected to

gel filtration chromatography by using Sephadex G-75 column. Total activity and

protein content in 5 ml of Sephadex G-75 fraction were 250 EU and 5 mg

respectively. The purified enzyme eluted as a single peak on Sephadex G-75 column

had a specific activity of 50 EU/mg. An overall 71.37 % fold purification and specific

activity of 50.0 EU/mg protein was achieved with 35.71% recovery (Table 21). The

fraction from Sephadex G-75 column was subjected to SDS-PAGE to determine

purity of enzyme.

Table 21: Summary of purification of Ammonium monooxygenase enzyme

Purification Step Volume

(ml)

Total

Activity

(EU)

Total

Protein

(mg)

Specific

Activity

(EU/mg )

Recovery

(%)

Fold

Purification

Culture Filtrate 120 700 900 0.7 100 1

(NH4 )2SO4

Fraction

40 550 140 3.9 78.57 5.57

DEAE- Sepharose

CL6B Fraction

14 400 29 13.8 57.14 19.70

Sephadex

G-75 Fraction

5 250 5 50 35.71 71.37

Values represented in the table are means of three separately conducted experiments.

157

4.8.1b. Determination of molecular weight of Ammonium monooxygenase

(AMO) enzyme

The molecular mass of Ammonium monooxygenase enzyme of B.cereus

was determined by SDS-PAGE. The appearance of two seperated bands on SDS-

PAGE suggested that the enzyme was purified. The purified AMO contained two

polypeptides of molecular masses nearer to 36 and 42 kDa., when compared to the

standard molecular weights of electrophoresis by using SDS-PAGE (Fig 30).

4.8.2. Partial Purification and characterization of Nitrite oxidoreductase (NOR)

enzyme:

4.8.2a. Purification:

The cell free culture filtrate obtained by sonication which was suspended in

10mM Potassium Phosphate Buffer (KPB) was used as crude enzyme source. The

crude enzyme was first precipitated with 80% ammonium sulfate saturation and a

two-step chromatography was conducted to purify the NOR enzyme. DEAE-

cellulose column chromatography had purified the enzyme partially. The final

purification step i.e., Gel filtration chromatography (Sephadex-75) yielded pure NOR.

The results of purification were summarized (Table 22). In order to check

purification, aliquots of different fractions were located and run on SDS-PAGE. The

purification fold was 9.06 with an yield of 31.4%. Its specific activity was 3.1 EU/mg.

158

Table 22: Summary of purification of Nitrite oxidoreductase enzyme

Purification

Step

Total

Volume

(ml)

Total

Protein

(mg)

Activity

(n mole/min)

Specific

Activity(U/mg)

Yield

(100%)

Purification

fold

Crude Enzyme 10 2.6 0.89 0.342 100 1

Ammonium

Sulphate

precipitation

8 0.68 0.6 0.882 67.4 2.578

DEAE

Cellulose

6 0.59 0.39 0.661 43.8 1.932

Sephadex G-75 1 0.09 0.28 3.1 31.4 9.06

4.8.2b. Determination of molecular weight of Nitrite oxidoreductase enzyme:

The molecular mass of Nitrite oxidoreductase of B. cereus was determined by

SDS-PAGE. The appearance of a single band on SDS-PAGE suggested that the

enzyme was purified and monomeric. It revealed a single band with molecular weight

of approximately 63 kDa, when compared to the standard molecular weights of

electrophoresis by using SDS-PAGE (Fig 31).

4.9. Effect of Nitrifying Bacillus cereus on growth of groundnut variety JL-24

Plant growth promotion as influenced by inoculation with nitrifying bacteria

was measured for groundnut plants variety JL 24 during 15 and 40 days of growth

interms of shoot length, root length, no.of leaves and total dry mass over uninoculated

controls. The following treatments were set up with different variables to evaluate the

inoculation effect with seed treatment and NO3- - N availability.

T0: Water (negative control), T1: Nitrogen (positive control), T2: NPK

(Fertilizer control), T3: NPK ( N-0, P-65.5 mg, K- 167 mg) + Bacterial inoculum, T4:

NPK ( N- 180 mg, P-65.5 mg, K- 167 mg) + Bacterial inoculum and T5: NPK ( N-

360 mg, P-65.5 mg, K- 167 mg) + Bacterial inoculum.

159

The plant growth promotion in terms of shoot length, root length, no.of leaves

and total dry weight of plant were observed significantly high for the treatments T5,

T4, T3 compared to the controls both at 15th

and 40th

day of plant growth as

influenced by inoculation with nitrifying bacteria (Fig 32). Significant results were

obtained for plant growth with the treatment T5 (Table 23 & 24).

Table 23: Effect of Nitrifying bacteria on plant growth promotion of Groundnut

JL-24 on 15th

Day.

S.No Seed

Treatment

% Seed

germination

No.of

leaves

Root

length

(cm)

Shoot

length

(cm)

Height

Root +

Shoot

(cm)

Weight

(gms)

Seedling

index

1 T0 70 8 10 22 32 5.20 2240

2 T1 100 13 15 25 40 6.50 4000

3 T2 100 16 17 23 33 5.70 3300

4 T3 100 15 13 22 35 5.80 3500

5 T4 100 18 19 28 47 7.25 4700

6 T5 100 22 21 30 51 10.12 5100

Table 24: Effect of Nitrifying bacteria on plant growth promotion of Groundnut

JL-24 on 40th

Day.

S.No Seed

Treatment

% Seed

germination

No.of

leaves

Root

length

(cm)

Shoot

length

(cm)

Height

Root +

Shoot

(cm)

Weight

(gms)

Seedling

index

1 T0 70 16 12 27 39 9.10 2730

2 T1 100 21 18 29 47 11.25 4700

3 T2 100 26 21 31 52 13.78 5200

4 T3 100 25 20 31 51 13.30 5100

5 T4 100 28 24 34 58 17.28 5800

6 T5 100 31 26 37 63 24.22 6300

The results in tables 23 and 24 had clearly shown significant increase in the

shoot length (30, 37 cm); root length (21, 26 cm) and 10.12, 24.22 gms of dry weight

per plant respectively during 15 and 40th

day of plant growth with the treatment T5.

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