ENHANCING BIOAVAILABLE PHOSPHOROUS IN SOIL THROUGH …

ENHANCING BIOAVAILABLE PHOSPHOROUS IN SOIL

THROUGH SULFUR OXIDATION BY THIOBACILLUS

IRFAN ULLAH

09-arid-1647

Department of Soil Science & Soil and Water Conservation Faculty of Crop and Food Sciences

Pir Mehr Ali Shah Arid Agriculture University Rawalpindi

Pakistan 2013

ii

ENHANCING BIOAVAILABLE PHOSPHOROUS IN SOIL

THROUGH SULFUR OXIDATION BY THIOBACILLUS

by

IRFAN ULLAH

(09-arid-1647)

A thesis submitted in partial fulfillment of

the requirement for the degree of

DOCTOR OF PHILOSOPHY

in

SOIL SCIENCE

Department of Soil Science & Soil and Water Conservation Faculty of Crop and Food Sciences

Pir Mehr Ali Shah Arid Agriculture University Rawalpindi,

Pakistan 2013

iii

CERTIFICATION

I hereby undertake that this research is an original one and no part of

this thesis falls under plagiarism. If found otherwise, at any stage, I will be

responsible for the consequences.

Student’s Name: IRFAN ULLAH Signature: ________________

Registration No: 09-arid-1647 Date: _________________

Certified that the contents and form of thesis entitled "ENHANCING

BIOAVAILABLE PHOSPHOROUS IN SOIL THROUGH SULFUR

OXIDATION BY THIOBACILLUS" submitted by Mr. Irfan Ullah have

been found satisfactory for the requirement of the degree.

Supervisor _________________________ (Dr. Ghulam Jilani) Member _________________________ (Dr. Khalid Saifullah Khan) Member _________________________ (Dr. Muhammad Rasheed) Chairperson: ________________________

Dean: ______________________________

Director Advanced Studies: _______________________

iv

v

DEDICATION

I dedicate this work to my parents who are no more with

me to see their prayers come true

vi

CONTENTS

Page

Acknowledgements xvi

Abstract xvii

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 4

2.1 Soil Phosphorous 4

2.2 Soil Phosphorous Fractions 5

2.3 Microbial Effect on Soil Phosphorous and Crop Yield 7

2.4 Soil Sulfur 8

2.5 Biological Sulfur Oxidation in Soil 15

3. MATERIALS AND METHODS 22

3.1 Screening of Sulfur Oxidizing Bacteria from Different Microbial

Ecologies

22

3.1.1 Isolation of Sulfur Oxidizing Bacteria 22

3.1.2 Screening of Sulfur Oxidizing Bacteria for Phosphorous

Solubilization

23

3.1.3 Biochemical Characterization of Selected Isolates 26

3.2 Comparative Efficiency of the Isolates for Soil Phosphorous

Solubilization

29

3.3 Sulfur Oxidizing Bacteria Application on Plant Growth and

Phosphorous Uptake

29

3.4 Plant Analyses 32

3.5 Soil Sampling and Analyses 33

3.5.1 Phosphorous Fractionation 35

3.6 Statistical Analyses 40

4. RESULTS AND DISCUSSION 41

4.1 Screening of Sulfur Oxidizing Bacteria from Different

Microbial Ecologies

41

vii

4.1.1 Occurrence of Sulfur Oxidizing Bacteria 41

4.1.2 Screening of Sulfur Oxidizing Bacteria for Phosphorous

Solubilization

41

4.1.2.1 The pH reduction 41

4.1.2.2 Colour change 45

4.1.2.3 Phosphorous solubilization index 46

4.1.2.4 Phosphorous solubilization efficiency 47

4.1.3 Characterization and Identification of Sulfur Oxidizing Bacteria 63

4.2 Comparative Efficiency of the Isolates for Soil Phosphorous

Solubilization

66

4.2.1 Basic Soil Analyses before Incubation 66

4.2.2 Changes in Basic Soil Analysis during Incubation 66

4.2.3 Soil Phosphorous Fractionation before Incubation 72

4.2.4 Changes in Soil Phosphorous Fractionation during Incubation 74

4.2.5 Phosphorous Bioavailability Enhancement during Incubation 86

4.2.6 Soil Sulfur Fractionation before Incubation 88

4.2.7 Changes in Soil Sulfur Fractionation during Incubation 88

4.2.8 Micronutrients Bioavailability Enhancement during Incubation 93

4.2.9 Interrelationship among Various Soil Variables 97

4.2.10 Interdependence among Various Soil Phosphorous Fractions 105

4.3 Sulfur Oxidizing Bacteria Application on Plant Growth and

Phosphorous Uptake

111

4.3.1 Basic Soil Analyses of Rice and Wheat Field 111

4.3.2 Treatment Effect on Bio-available Phosphorous contents at

Rice Harvest

111

4.3.3 Treatment Effect on Growth and Yield Attributes of Rice 114

4.3.4 Phosphorous Uptake by Rice 119

4.3.5 Correlation among Various Soil and Rice Variables 119

4.3.6 Treatment Effect on Bio-available Phosphorous Contents at

Wheat Harvest

124

viii

4.3.7 Treatment Effect on Growth and Yield Attributes of Wheat 128

4.3.8 Phosphorous Uptake by Wheat 129

4.3.9 Correlation among Various Soil and Wheat Variables 131

4.3.10 Basic soil analyses for Maize Field 137

4.3.11 Treatment Effect on Soil Bio-available Phosphorous Contents at

Maize Harvest

137

4.3.12 Treatment Effect on Growth Parameters of Maize 143

4.3.13 Treatment Effect on Yield Attributes of Maize 144

4.3.14 Phosphorous Uptake by Maize 145

4.3.15 Correlation among Various Soil and Maize Variables 148

SUMMERY 155

CONCLUSIONS 157

LITERATURE CITED 158

APPENDICES 200

ix

List of Tables

Table No.

Page

1. Ecology-wise description of sulfur oxidizing bacteria 42

2. The pH reduction by sulfur oxidizing bacteria in tricalcium phosphate

media during the incubation period

48

3. Sulfates production by sulfur oxidizing bacteria in tricalcium

phosphate media during incubation period

49

4. Phosphorous solubilization by sulfur oxidizing bacteria in tricalcium


51

5. Correlation among various variables in tricalcium phosphate media 53

6. The pH reduction by sulfur oxidizing bacteria in rock phosphate

leach suspension

56

7. Sulfates production by sulfur oxidizing bacteria in rock phosphate

leach suspension

57

8. Phosphorous solubilization by sulfur oxidizing bacteria in rock

phosphate media

59

9. Correlation among various variables in rock phosphate media 61

10. Biochemical characterization of the isolates 64

11. Basic soil analyses before incubation 67

12. Changes in soil pH during incubation 68

13. Soil electrical conductivity during incubation 70

14. Changes in soil CaCO3 contents during incubation 71

15. Soil phosphorous fractionation before incubation 73

16. Soil phosphorous fractions after 30 days of incubation 75



19. Enhancement in soil phosphorous bioavailability during incubation 85

20. Soil sulfur fractionation before incubation 87

21. Changes in sulfur fractions 30 days after incubation 89

x



24. Soil micronutrients contents after 30 days of incubation 94



27. Correlation among various soil variables 98

28. Linear regression analyses among various soil variables 100

29. Interrelationship among various soil phosphorous fractions 103

30. Basic soil analyses before rice-wheat experiment 112

31. Soil bio-available phosphorous contents at rice harvest 113

32. Treatment effect on growth parameters of rice 115

33. Treatment effect on yield attributes of rice 116

34. Phosphorous uptake by rice 118

35. Correlation among various rice variables 120

36. Linear regression analyses among soil and rice variables 121

37. Soil bio-available phosphorous contents at wheat harvest 125

38. Treatment effect on growth parameters of wheat 126

39. Treatment effect on yield attributes of wheat 127

40. Phosphorous uptake by wheat 130

41. Correlation among various wheat variables 132

42. Linear regression analyses among soil and wheat variables 133

43. Basic soil analysis before spring-autumn maize experiment 138

44. Soil bio-available phosphorous contents at maize harvest 139

45. Treatment effect on growth parameters of maize 141

46. Treatment effect on yield attributes of maize 142

47. Phosphorous uptake by spring maize 146

48. Phosphorous uptake by autumn maize 147

49. Correlation among various maize attributes 153

50. Linear regression analyses among soil and maize variables 154

xi

List of Figures

Figure No.

Page

1. Schematic diagram of enhancing bio-available P in soil through sulfur

oxidation by Thiobacilli

17

2. Frequency of SOB in sampling ecologies indicating highest number

different SOB in industrial waste water

43

3. Phosphorous solubilization relation with pH in tricalcium phosphate

media changes: a to d represent data from day 08, 16, 24 and 32,

respectively

54

4. Relation of P solubilization with pH in tricalcium phosphate leach

suspension changes: a to d represent data from day 10, 20, 30 and 40,

respectively

62

5. Soil pH relation with (a) ECe and (b) CaCO3 contents changes during

incubation: a1 to a3 and b1 to b3 represent data from day 30, 60 and

90, respectively

80

6. Relation of Ca2-P with (a) soil pH and (b) CaCO3 contents changes

during incubation: a1 to a3 and b1 to b3 represent data from day 30, 60

and 90, respectively

81

7. Bio-available P relation with (a) soil pH and (b) CaCO3 contents

changes during incubation: a1 to a3 and b1 to b3 represent data from

day 30, 60 and 90, respectively

82

8. Relations of Ca8-P with (a) soil pH and (b) CaCO3 contents changes



83

9. Ca10-P relation with (a) soil pH and (b) CaCO3 contents changes



84

xii

10. Zinc extractable relation with soil pH (a) and CaCO3 (b) changes



106

11. Relation of iron extractable with soil pH (a) and CaCO3 (b) changes



107

12. Manganese extractable relation with pH (a) and CaCO3 (b) changes



108

13. Relation of copper extractable with pH (a) and CaCO3 (b) changes



109

14. Boron extractable relation with pH (a) and CaCO3 (b) changes during

incubation: a1 to a3 and b1 to b3 represent data from day 30, 60 and

90, respectively

110

15. Soil bio-available P relation with different rice yield attributes 122

16. Phosphorous uptake relation with different rice yield attributes 123

17. Soil bio-available P relation with different wheat yield attributes 134

18. Phosphorous uptake relation with different wheat yield attributes 135

19. Soil bio-available P relation with different spring maize yield attributes 149

20. Relation of P uptake with different spring maize yield attributes 150

21. Soil bio-available P relation with different autumn maize yield

attributes

151

22. Phosphorous uptake relation with different autumn maize yield

attributes

152

xiii

List of Plates

Plate No. Page

1. Isolation and purification of sulfur oxidizing bacteria

44

2. Response of rice and wheat to Thiobacillus

136

xiv

List of Appendices

App. No. Page

1. Description of samples for the isolation of SOB 200

2. The pH reduction by SOB isolates 207

3. Colour change by SOB isolates 210

4. Phosphorous solubilization index of SOB

211

xv

Abbreviations Used Abbreviations Description

BAP Bio-available phosphorous

BY Biological yield

CW Canal water

ECe Electrical conductivity of saturated soil paste extract

EDTA Ethylene-di-amime-tctra acetic acid

H1 Harvest index

IS Industrial waste sludge

IW Industrial wastewater

MGY Maize grain yield

MR Maize rhizosphere

MSY Maize stalk yield

PF Paddy fields

PSI Phosphorous solubilization index

PY Paddy yield

r Regression coefficient

RP Rock phosphate

SM Sulfur mud

SOB Sulfur oxidizing bacteria

SR Sugarcane rhizosphere

SS Sewage sludge

SW Sewage water

SY Straw yield

TCP Tricalcium phosphate

TPU Total phosphorous uptake

WGY Wheat grain yield

WR Wheat rhizosphere

WSY Wheat straw yield

xvi

ACKNOWLEDGEMENTS

All the praises and thanks to Almighty ALLAH, the compassionate, the

merciful and the only creator of the Universe, Who bestowed me with the

potential and ability to contribute a little to the existing ocean of knowledge. I

offer my humblest thanks to the Holy Prophet Hazarat MUHAMMAD (Sallallaho-

AIlehe-Wasalam) who is forever a torch of guidance and knowledge for me.

1 would like to express my profound gratitude to my supervisor Dr.

Ghulam Jilani, Associate Professor, Dr. Khalid Saifullah Khan, Associate

Professor, Dr. Mohammad Saleem Akhtar, Professor and Chairman, Department

of Soil Science & SWC and Dr. Muhammad Rasheed, Assistant Professor,

Department of Agronomy for their continuous and critical assessments, inspiring

guidance and helpful suggestions in the conduct of this work.

Special thanks and appreciations are extended to Mr. Jamshed Iqbal

Cheema, Chief Executive, Muhammad Azam Cheema, Director A & F, Dr.

Muhammad Irfan-Ul-Haq, General Manager (Technical) and Mr. Anwer Khan,

Microbiologist for providing research facilities and valuable suggestions during

my research work at Auriga Research Station, Lahore.

1 wish to record my sense of admiration for my colleagues Syed Sajjad

Hussain Bokhari, Ch. Khalil Ahmed, Syed Sajjad Haider Kazmi, Muhammad

Ather Latif, Lubna Ayub Durrani and Ijaz Ahmed for their sincere co-operation.

Words are lacking to express humble obligation to my family members

particularly devoted wife, affectionate brother (Dr. Rizwan Ullah Warraich),

father-in-law (Ch. Shafique Ahmed Cheema), uncles (Ch. Riaz Ahmed Warraich

and Ch. Mazher Ullah Warraich), brother-in-law (Ch. Hassan Ashraf Gill), sister,

sons (Muhammad Arslan Ullah Warraich and Muhammad Salman Ullah

Warraich) and daughters for their sacrifices and offering countless prayers.

At last but not the least, this achievement is due to prayers and sacrifices of

my late father (Ch. Ehsan Ullah Warraich) and late mother, may Allah bless them

a place in the heaven and brightened their graves (Aameen).

(IRFAN ULLAH WARRAICH)

xvii

ABSTRACT

Phosphorus (P) fertilizer use efficiency is only 15 % in calcareous alkaline

soils as in Pakistan. Sulfur oxidizing bacteria (SOB) especially Thiobacillus spp.

solubilize the unavailable P in soil by synthesizing sulfuric acid. This study was

performed in three steps: (i) screening of SOB from different microbial ecologies,

(ii) use of different SOB with S levels for enhancing bio-available P, and (iii)

effect of SOB along with P fertilizer and S on plant growth and yield.

Sulfur oxidizing bacteria were isolated, screened, identified and the most

efficient SOB were found as the genus Thiobacillus in sulfur based ecologies such

as industrial wastewater, sewerage water and sulfur mud. These SOB isolates were

IW1, SW2, SS1, IW13, IW14, IW16 and SM1 which reduced the pH of different

media (thiosulphate, tricalcium phosphate and rock phosphate) in 07 to 40 days

after inoculation.

Four Thiobacillus isolates viz., IW16, SW2, IW1 and IW14 were

inoculated in soil with three S levels 50, 37.5 and 25 mg kg-1. Thiobacillus spp.

IW16 and SW2 reduced soil pH with 50 mg S kg-1 from 7.90 to 7.12 and 7.28

respectively. Similarly, Thiobacillus strain IW16 in combination with 50 mg S

kg-1 reduced CaCO3 contents from 7.14 to 6.93 % and solubilized Ca8-P and Ca10-

P. Increase in the concentration of sparingly soluble Ca2-P (20.33 mg kg-1) and

xviii

bioavailable P contents (net increase of 22.26 mg kg-1) were also recorded as a

result of P solubilization phenomenon.

Lastly field experiments were conducted on two permanent lay outs (plot

size 3 m × 3 m) at two different places growing for rice-wheat and maize-maize

crops. The best Thiobacillus isolates (IW16 and SW2) along with 100 kg S ha-1

were inoculated in combination with two doses of P fertilizer viz., 45 and 90 kg

P2O5 ha-1. Significant increase in the concentration of bio-available P in soil was

recorded through bacterial S oxidation in both experiments. Growth and yield

parameters of the tested crops (rice, wheat and maize) exhibited positive

significant correlation with P solubilization through S oxidation by Thiobacilli

with the highest values by strain IW16 along with 100 kg S ha-1. Interaction

between Thiobacillus spp. and S was highly significant in enhancing the growth

and yield of crops. Treatment of soil with Thiobacilli and S was the best practice

for enhancing bioavailability of P already present as fixed P in huge quantity.

Chapter 1

INTRODUCTION

An adequate phosphorous (P) supply in rhizosphere is essential to activate

plant roots for P uptake essential for plant growth. Total P in cultivated soils

ranges from 400-3000 mg kg-1 (Richardson et al., 2005), and the bio-available P is

as low as 1.0 mg kg-1 (Goldstein, 1994; Vassilev et al., 2001). Fertilizer P is

converted to unavailable forms which reduces P fertilizer use efficiency to as low

as 10 to 25 % (Khiari and Parent, 2005).

Better understanding on soil P cycle and P fractions in relation to plant

growth is essential for better P management, enhancing bio-available P and

decreasing fertilizer use (Shen et al., 2011). Main factors for P fixation in acidic

soils are oxides and hydroxides of iron and aluminum while in calcareous soils

major cause is the high amount of CaCO3 (Pant and Warman, 2000). Majority of

Pakistani soils are alkaline and calcareous in nature with pH > 7.5 and CaCO3 >

7.0 % (Ahmad et al., 2006). More than 90 % soils are moderate to high P deficient

(Nisar et al., 1992). Phosphorous fixation is a concern in alkaline and calcareous

soils (Huang, 1998). Phosphorous dynamics in calcareous soils depends on CaCO3

and clay contents (Tisdale et al., 2002). A chain of fixation reactions starts after

the addition of P fertilizers in these soils. These reactions range from surface P

adsorption on lime and clay surfaces to the precipitation of different Ca-P

1

2

compounds (Leytem and Mikkelsen, 2005) resulting in very slow release of P into

soil solution.

Sulfer oxidizing bacteria (SOB) oxidize elemental S to sulfates through

biological oxidation process (Pokorna et al., 2007; Briand et al., 1999). Among

SOB the genus Acidithiobacillus is more effective for biological S oxidation in

soil (Yang et al., 2010). Biological S oxidation produces acidity which increases

the solubility of plant nutrients including P (Stamford et al., 2003; El-Tarabily et

al., 2006; Yang et al., 2010). Elemental S is effective for enhancing P

bioavailability in soil through the process of S oxidation (Aria et al., 2010). The

bacterial S oxidation produces sulfuric acid, which results in P release from rock

phosphate (Besharati et al., 2007; Chi et al., 2007). Rock phosphate solubilization

has positive correlation with the quantity of sulfuric acid generated (Bhatti and

Yawar, 2010; Yang et al., 2010).

Several studies have reported soil P solubilization with different

amendments (Stamford et al., 2003): application of S (Rajan, 1983; Garcia et al.,

2007), use of organic matter (Nishanth and Biswas, 2008) and inoculation with

bacteria (Chi et al., 2007). Calcareous soils contain insoluble calcium phosphates

viz., apatite (Ca5(PO4)3(OH,F,Cl) and calcite (CaCO3) surface sorbed P. Sulfuric

acid produced by SOB through biological S oxidation reacts with the insoluble

calcium bounded P minerals and brings it to plant available P forms. The

3

ecologies where the most efficient P-solubilizing SOB could be found remain to

be explored.

Phosphorous fractionation helps in better understanding of different P

pools in the soil system (Nair et al., 1995; Hinsinger, 2001). Therefore, it is very

important to determine the relationship between different inorganic fractions of P

in soil and their solubilization behavior through interactive effect of Thiobacilli

and S application. Furthermore, the effect of P solubilization and enhancement

through S oxidation by Thiobacillus spp. on different plant growth and yield

parameters is also very critical.

This study examined the role of SOB with S for enhancing P availability in

soil. The objectives were (i) to isolate efficient SOB isolates for enhancing bio-

available P in soil, (ii) to find out the appropriate rate of S for enhancement of bio-

available P, and (iii) to find out the best combination of P fertilizer and S with

SOB for the enhancement of bio-available P in soil.

4

Chapter 2

REVIEW OF LITERATURE

A review of previous studies on role of sulfur oxidizing bacteria for P

solubilization and effect on crop production is presented here under:

2.1 SOIL PHOSPHOROUS

Total soil P ranges between 200 to 3000 mg kg-1 (Richardson et al., 2005),

and less than 1 % is available to the plants. Two major forms of soil P are (i)

organic P and (ii) inorganic P which exist in variety of compounds equilibrium

with each other. These compounds range in solubility from soluble P to extremely

stable P (Turner et al., 2007). Soil inorganic P constitutes 50 to 75 % of the total

P, and exists as compounds of calcium, iron and aluminum. Phosphorous

transformations in soil are affected by microbial activities, plant species, and the

nature of soil, environment and soil cultivation (Wright, 2009).

Primary P minerals (strengite, apatites and variscite) which are highly

stable and the secondary P precipitates of calcium, aluminum and iron have

different solubility rates (Oelkers and Valsami-Jones, 2008). Phosphorous makes

complexes with surfaces of aluminum / iron oxides and clay minerals, while inner

sphere complex of protonated bidentate complex is leading in acidic soils (Arai

and Sparks, 2007).

4

5

Phosphorous dynamics in calcareous soils is linked with Ca-phosphate

precipitation and adsorption on soil colloids responsible for P retention (Nisar et

al., 1999; Delgado et al., 2002). Precipitation is dominated in slightly calcareous

to high calcareous soils (Lindsay et al., 1989). Adsorption of phosphorous occurs

on clay minerals (Devau et al., 2010) and calcium carbonate surfaces (Larsen,

1967). Precipitation starts with formation of di-calcium phosphate (DCP) which is

sparingly soluble and available to plants, is further converted into more stable

octa-calcium and hydroxy-apatite (Arai and Sparks, 2007).

Soil organic P occurs as phosphonates and inositol phosphates with active

forms as monoesters, di-esters, orthophosphate and polyphosphates (Turner et al.,

2002; Condron et al., 2005). Mineralization of organic P, facilited by soil

microorganisms and plant root secretions is important in dynamics of soil P and its

availability to plants (Richardson, 2001). Phosphorous solubilizing bacteria

solubilize soil P by acidifying rhizosphere through the production of organic acids

and enzyme like phytases and phosphatases (Chen et al., 2006; Richardson et al.,

2009; Jones and Oburger, 2011).

2.2 SOIL PHOSPHOROUS FRACTIONS

Mostashari et al. (2008) reported that calcareous soils have total P from

700 to 1040 mg kg-1. They further recorded Ca2-P from 1.6 to 42.3 mg kg-1, Ca8-P

from 72 to 314 mg kg-1, Al-P from 14.5 to 54.8 mg kg-1, Fe-P from 8.4 to 34.8 mg

kg-1, O-P from 5.9 to 33.4 mg kg-1 and Ca10-P from 262 to 697 mg kg-1. They also

6

found a positive correlation of Olsen-P with soluble-P, di-calcium-P, iron-P and

occluded-P. All P-fractions in the soil had a positive correlation among them and

showed a dynamic relationship. Ahmad et al. (2006) determined total soil P

between 652 to 1245 mg kg-1soil after taking 24 soil samples from the area of

University of Agricultural Faisalabad, Pakistan. Memon et al. (2011) reported that

apatite P fraction is the biggest (500 to 600 mg kg-1 in alluvial soils and 200 to 300

mg kg-1 in loess and shale derived soils) amongst the entire P fraction in the soils

of Indus plain (Pakistan).

Adhami et al. (2007) estimated different P fractions in soil and reported

their sequence as Ca2-P<Fe-P<Al-P<O-P<Ca8-P<Ca10-P. These forms showed

positive correlation with exchangeable-P and Olsen-P. Adhami et al. (2006)

recorded a considerable association between Ca-bound P and soil silt contents and

non significant correlation between calcium bounded and iron bounded P.

However, Fan et al. (2007) found a negative correlation between clay contents and

Olsen-P, silt contents and Ca8-P while they recorded positive correlation among

occluded-P, Al-P, Fe-P, solution P and total P. Delgado et al. (2000) concluded

that plants utilize water soluble P for their growth and development which is

usually estimated by Olsen method in calcareous soils. Calcareous soils in

Western Australia contain 35 % organic P which has positive correlation with Fe-

P fraction and organic matter.

7

Quantities of different soil inorganic fractions of P reported by Samadi

(2003) for Ca2-P, Ca8-P, Al-P, Fe-P, occluded-P, Ca10-P were 15, 27, 26, 14, 20

and 16 mg kg-1, respectively. Addition of organic P fertilizers in soil increases

microbial activities which results in the conversion of plant unavailable P forms to

plant available inorganic P fractions (Lee et al., 2004). Turan et al. (2007) studied

microbial effect on different soil P fractions using tomato as a test crop. They

recorded a significant increase in plant available P fractions in soil and increase in

shoot and root dry weight of tomato plants under the effect of PSB.

Different P fractionation methods are reported (Condron and Newman,

2011; Yang and Post, 2011) and some more appropriate for calcareous soils. Jiang

and Gu (1989) P fractionation procedure is particular for calcareous soils wherein

three calcium phosphate forms are identified as: (i) dicalcium phosphate (ii) octa-

calcium phosphate, and (iii) apatite.

2.3 MICROBIAL EFFECT ON SOIL PHOSPHOROUS AND CROP

YIELD

Yazdani et al. (2009) noted an increase in bio-available P level in soil

through phosphorous solubilizing microorganisms (PSM) which increased maize

straw yield. Further, Plant dry matter, total P concentration in plants and plant P

uptake has positive correlation with the amount of available P in soil (Alam and

Shah, 2002; Singh et al., 2002). Similarly, plant biomass increases at high level of

Olsen P in the soil (Afzal and Bano, 2008). Growth and yield attributes of maize

8

crop viz., number of cobs plant-1, number of grains cob-1, grain weight cob-1and

grain yield have positive significant relationship with the quantity of plant

available P in soil (Khan et al., 2005; Rahman, et al., 2007). Likewise, several

scientists (Maqsood et al., 2001; Hussain et al., 2006) reported positive significant

effect of bio-available P on maize grain yield.

Panhwar et al. (2011) recorded an increase in rice yield with increase in

plant available P concentration in soil under the influence of PSB. High bio-

available P levels in soil significantly increased yield parameters of wheat crop

(Kaleem et al., 2009; Rahman, et al., 2011). Sultani et al. (2004) determined high

P uptake by wheat plants with high quantity of plant available P in soil. Yadav et

al. (2011) recorded maximum yield attributes of wheat crop when PSB were

inoculated with P fertilizer.

2.4 SOIL SULFUR

Important form of soil sulfur (S) is sulfate and its concentration in soil is

below 5 % of the total soil S. This SO42--S is further categorized into two forms

viz., soil solution sulfate and adsorbed sulfate (Barber, 1995). But in case of

calcareous soils sulfate is also present in precipitated form with Ca / Mg (Tisdale

et al., 1993). Hu et al. (2005) studied SO42--S in 64 calcareous soils and observed

that its amount is positively correlated with CaCO3 contents in soil. Sulfate-S is

precipitated with CaCO3 upto 42 % in calcareous soils of Canada (Roberts and

Bettany, 1985). Some non calcareous soils also precipitate SO42--S with CaCO3

9

(Chen et al., 1997). Morche (2008) stated that the major part of 1N HCl extracted

S from soils with < 1 % CaCO3 is organic bound S.

Soil solution contains small amount of sulfates. This concentration remains

changing and is associated with plant uptake, application of S-fertilizers,

immobilization and mineralization (McLaren and Cameron, 2004). High quantity

of of sulfates in upper soil surface depends on addition of S-fertilizers (Eriksen et

al., 1995) and organic S mineralization (McLaren and Cameron, 2004). In winter

and spring seasons of sulfates concentration has the lowest values due to minimum

mineralization rate (Ghani et al., 1990). Adsorbed of sulfates are also very

important and considered as a plant available source of S. Mechanism of S

adsorption is vital for restricting it from leaching (McLaren and Cameron, 2004).

Rahman et al. (2011) reported that elemental S dropped pH of the calcareous soils

from 9.08 to 7.56 and played a key role in reducing CaCO3 contents and thus

enhanced the uptake of plant nutrients such as P, S and micronutrients. Kaya et al.

(2009) also considered elemental S vital for lowering soil pH and making plant

nutrients available in calcareous soils.

Organic S is the major fraction of S in soil (Yang et al., 2007b). Its

concentration in surface soils is upto 95 % (Kertesz and Mirlau, 2004) and it may

go upto 98 % of the total sulfur in soil (Bloem, 1998). Organic S compounds exist

in animals, plants and microorganisms and present in soil before or after the

death/decay of animals, plants and microorganisms (Tabatabai and Bremner,

10

1972). Very limited information is available about the chemical identity of organic

S compounds as they are present as heterogeneous mixture (Kertesz and Mirleau,

2004). However, the quantity of organic S compounds in soil depends upon the

amount of organic C and total N (Wang et al., 2006).

Two major groups can be differentiated among the variety of S-compounds

viz., (i) ester sulfates and (ii) C-bonded S. Some more soil S-compounds like

heterocyclic S and sulfonates are also present (Kertesz and Mirleau, 2004), but

these are less important (Edwards, 1998). Ester sulfates (C-O-S) are estimated by

the conventional method of S fractionation through the reduction of hydriodic acid

(HI) as described by Shan and Chen 1995. Then ester sulfate is deducted from

total S to calculate C-bonded S. Ester sulfates consist of various compounds like

sulfates, phenolic sulfates, sulfated polysaccharides and choline (Edwards, 1998)

constituting organic S from 27 to 45 % and their amount differs from 52 to 92 mg

kg-1 soil (Eriksen et al., 1995). Addition of farmyard manure increases C-bonded S

in the soil (Forster et al., 2012).

Sulfur cycle in soils revolves between organic and inorganic forms.

Immobilization of inorganic S results in organic S compounds which are inter-

transferable and mineralization converts immobilized S to plant available

inorganic S fractions (Kertesz and Mirleau, 2004). These two processes are

controlled by microorganisms (Ghani et al. 1992). Organic S is very important in

providing SO42--S to the plants after passing through the process of mineralization

11

(Eriksen, 1997). Microbial activity is essential for the mineralization of C bonded

S compounds (Jaggi et al., 1999). It has been noted that ester sulfates mineralize

easily as compared to C-bonded S compounds (Kertesz and Mirleau, 2004).

Oxidation of C-bonded S converts it into ester sulfates which after mineralization

transfer to SO42- (Ghani et al., 1991). Soil capacity to supply S to plants depends

on the composition of organic-S in soil (Yang et al., 2007b).

Mechanisms of S mineralization and immobilization are taking place in

soil at the same time (Maynard, 1982) resulting in release and fixation of

inorganic SO42-. Whole S cycle consists of many processes (Eriksen et al., 1995)

and hence S bioavailability in soil depends on net S-mineralization (Dedourge et

al., 2003). These two processes (mineralization and immobilization of S) are

affected by soil temperature, soil moisture, application of S fertilizers into the soil,

soil organic matter contents, C:S ratio, atmospheric-S-inputs and nature of plants

present in the soil (Edwards, 1998; Scherer, 2001). Organic S mineralizes annually

@ 3.3 to 6.7 μg S g-1 soil year-1, which is not sufficient to meet the crop

requirements (White, 2006).

Two inorganic S fractions are considered as plant available fractions, one

is water soluble S and other is adsorbed S, while some part of organic S

compounds that are easily mineralized also have importance in S availability

(Bettany et al., 1974). Estimation of plant available S is done by various methods

wherein different extractants are used such as H2O, CaCl2, KH2PO4, NaH2PO4,

12

and Ca(H2PO4)2 and NaHCO3 (Prietzel et al., 2001; Hu et al., 2005; Lehmann et

al., 2008). Matula (1999) stated that CaCl2 extractant may not be suitable due to

its precipitation reaction with calcium in calcareous soils.

Srinivasarao et al. (2004) determined different S fractions in Indian soils.

They reported total S from 241 to 391 mg kg-1, organic S from 191 to 362 mg kg-1,

adsorbed S from 12.9 to 59.0 mg kg-1 and available S from 3.5 to 9.2 mg kg-1. Hu

et al. (2005) concluded that organic S in calcareous soils was a dominant fraction

(>77 % of the total S). Total S concentration in soils generally ranges between

0.01 to 0.1 % (Balik et al., 2007; Morche, 2008) and organic S constitutes the

main portion of the total S in soil (Kertesz and Mirleau 2004, Yang et al., 2007a).

Sulfates present in the soil solution are easily removable and can be determined by

water extracts (Prietzel et al., 2001), while sorbed S can be estimated by P based

extractants in calcareous soils (Hue et al., 2005).

Balik et al. (2009) studied different inorganic and organic fractions of soil

S by adopting S fractionation scheme of Morche (2008). They reported water

soluble S from 14.4 to 29.8, sorbed S from 5.6 to 11.0, occluded S from 8.3 to

16.9, organic S from 99.2 to 228.0 and total S from 133.0 to 284.0 mg kg-1soil.

Sulfur fractionation scheme given by Morche (2008) was used in the present study

to observe changes in various S fractions in soil under the influence of S

application and sulfur oxidizing bacteria (SOB) inoculation.

13

Amount of inorganic soil S is very low as compared to the organic S. Main

inorganic S forms in soil are sulfates (SO42-) and sulfides (S). Aerobic soils

contain SO42- in sufficient amount (Bohn et al., 2001). Sulfites and elemental S in

soil may also present in small quantity (Watkinson and Kear, 1994).

Benchmark analysis of Pakistani soils revealed that 15 % soils of Pakistan

were S deficient (< 10 μg g-1), 30 % were satisfactory (11-30 μg g-1), 33 % soils

were found in the range of 31-99 μg g-1 and 22 % were in adequate range (> 100

μg g-1). Rainfed areas have low S contents than in irrigated areas. Canal and tube

well irrigated areas of central Punjab have the highest S concentration (> 100 μg

g-1) while well drained soils of Pothwar plateau had the lowest S contents (< 10 μg

g-1). Deficiency of S (< 10 μg g-1) was reported on 70 to 80 % area of Rawalpindi

district and Sindh province. Average S contents in Khyber Pakhtun Khwa

province are found 50 μg g-1 of soil. Average S conents in Baluchistan soils are

29 μg g-1 of soil. Deficiency of S was reported in different areas of Pakistan like

Pothwar (Punjab), Quetta-Pashin (Baluchistan) and Southern and Central Sindh

(Ahmed et al., 1994).

Sulfur is an important element in balanced plant nutrition and has

significant correlation with crop yield quantity and quality (Tiwari and Gupta,

2006; Gulati et al., 2007). Requirement of S for oil seed crops is more than fodder

crops. Sulfur deficiency in oil seed crops results in weak and less pod formation

and ultimately effects badly seed and oil production (Iqtidar and Jan, 2002). Sulfur

14

@ 30 to 60 kg ha-1 rendered positive effect on many crops under different

cropping systems on various Indian soil types (Hedge and Murthy, 2005). Singh et

al. (2002) found 17 to 30 % increase in crop yield as a result of S application.

Malhi and Gill (2002) observed significant increase in canola yield when S rate

increased from 15 to 30 kg ha-1.

Haneklaus et al. (1999) reported that 40 kg S ha-1 is sufficient in getting

maximum yield of oil seed rapes in North Germany. Choudhury et al. (2002)

found 40 % increase in rapeseed yield with 20 kg S ha-1. Application of S not only

enhances crop yield and crop quality but also it is economical as compared to N

and P fertilizers (Ghosh et al., 2000). Many studies showed that available S < 10

to 13 μg g-1 was deficient for majority of field crops (Hedge and Murthy, 2005)

and continuous S removal by crops results in S deficiency in soil (Aulakh, 2003).

Most of the S taken up by oil seed crops (70 to 80 %) reaches in seeds and

grains. Percentage of S translocation may be more in mustard and rapeseed

(Ghosh et al., 2000). Sufficient limit of S contents in canola is > 1 % S (Grant,

1991) while the critical limit of S for mustard crop is 0.21 to 0.24 % (Hedge and

Murthy, 2005). Bandyopadhyay and Chattopadhyay (2000) reported that the

average S contents in rapeseed plants were 0.10 % in India and Eriksen (2005)

reported 0.70 % in Denmark, while in Pakistan Ahmed et al. (1994) reported plant

S contents in raya crop between 0.94 to 1.11 %. Quantity of plant total S contents

and plant S uptake has a positive significant correlation with water soluble S

15

concentration in the soil solution (Tiwari and Gupta, 2006; Gulati et al., 2007,

Rahman, et al., 2011). Rahman et al. (2007) reported that biological and grain

yield of rice crop extensively enhanced with high quantity of water soluble S and

P in soil.

2.5 BIOLOGICAL SULFUR OXIDATION IN SOIL

The conversion of elemental S to sulfates in soil is necessary for its

availability to crops (Mahendra, 1988). Reduced form of S is sulfide of metals in

the soil. Its oxidation to sulfates takes place by sulfur oxidizing microorganisms.

Rate of S oxidation in coarse textured soils is faster and takes three to four weeks

for complete oxidation (Tandon, 1989). There is a close bacteria-substrate

interaction for oxidation of S (Briand et al., 1999). Basic substrate for SOB is

elemental S which oxidizes to sulfates during oxidation process (Pokorna et al.,

2007). Sulfur oxidation has two phases to predict S oxidation rate more suitably

viz., fast S oxidation and slow S oxidation phases (Slaton et al. 2001).

Current classification of SOB contains species of Thiobacillus,

Thiomicrospira and Thiosphaera. These are basically gram negative bacteria.

Some species of Xanthobacter, Paracoccus, Pseudomonas and Alcaligens are

heterotrophs and can also behave like chemolithotrophs by growing on inorganic

sulfur compounds (Kuenen and Beudeker, 1982).

16

Sulfur oxidizing bacteria include two main metabolic types, (i) obligate

chemolithotrophs which only depend on oxidizable sulfur compounds for growth

and (ii) heterotrophs that are also able to exercise the chemolithoautotrophic

mode. The species Thiobacillus thiooxidans, Thiobacillus ferrooxidans,

Thiobacillus thioparus, Thiobacillus neapolitanus, Thiobacillus denitrificans

(facultative denitrifier), Thiobacillu shalophilus (halophile) and few species of

Thiomicrospira are obligate chemolithotrophs. The species Thiobacillus

intermedius, Thiobacillus novellus, Thiobacillus acidophilus, Thiobacillus

aquaesulis, Paracoccus denitrificans, Xanthobacter tagetidis, P. versutus,

Thiomicrospira thyasirae, and Thiosphaera pantotroph are heterotrophs (Kelly

and Wood, 2000).

Thiobacilli generally enhances sulfur oxidation rate and it is further

boosted by the addition of sulfur in soil. Thiobacillus thiooxidans and Thiobacillus

ferrooxidans are mostly responsible for S oxidation in soils (Figure 1). They

utilize S to meet their energy requirement. Thiobacillus thioparus can not exist

below pH 2.5 (Masau et al., 2001). Large quantity of cations and anions can

inhibit the growth of T. thiooxidans (Suzuki et al., 1999). Therefore, S oxidation

may be affected by the amount of accumulated SO42- in soil (Harahuc et al., 2000).

Sulfur oxidation totally depends on SOB in many soils (Lawrence and Germida,

1991). Kelly and Wood (2000) reclassified genus Thiobacillus on the basis of

DNA tests, conditions for optimum growth such as pH, temperature (°C) and use

of energy source.

17

Soil P Bioavailable P

Inorganic & Organic

Figure 1. Schematic diagram of enhancing bio-available P in soil through sulfur

oxidation by Thiobacilli

S Oxidation Thioabacilli

Minerilzation / Solubilzation

S Reduction D.desulfuricans

Immobilization

18

Thiobacillus spp. are very important in the oxidization of S in soils (Yang

et al., 2010). The optimum temperature for biological S oxidation is from 30 to 40

◦C (Germida and Janzen, 1993). Sulfur oxidation produces sufficient amount of

sulfates like S fertilizers (Hassan et al., 2010; Aria et al., 2010; Yang et al., 2010).

Bacterial S oxidation takes place in the following oxidation steps:

Thiobacilli S° S2O3

2- S4O62- SO4

2-

Sulfur-oxidizing bacteria like Acidithiobacillus, Thiospirillopsis, and

Thiovulum under aerobic conditions oxidize inorganic S to sulfuric acid. The

reaction equation is as under:

S + H2O + 1.5O2 SO42- + 2H+, Δ Go = -587.1kJ / reaction.

Energy produced during the oxidation of S is used by SOB for the

synthesis of organic compounds from carbon dioxide (Madigan et al., 2003).

Bacterial sulfur oxidation is an acid generating phenomenon which decreases pH

of the media. Bacterial oxidation of S by A. thiooxidans and A. ferrooxidans is

shown by the following chemical equations:

S° + 1.5 O2 + H2O H2SO4

Fe3S4 + 7.5 O2 + H2O 3FeSO4 + H2SO4

Insoluble calcium bonded P compounds react with sulfuric acid produced

in the above reaction and resultantly gypsum is formed.

CaCO3 + H2SO4 + H2O CaSO4 ∙2H2O + CO2

19

Ca5 (PO4)3F + 5H2SO4 + 10H2O 3H3PO4 +

5CaSO4∙2H2O + HF

Phosphoric acid thus formed attacks on fluorapatite and P is dissolved and

converted to dicalcium phosphorous.

Ca5(PO4)3F+7H3PO4 5Ca(H2PO4)2 + HF

Bhatti and Yawar (2010) conducted an experiment by using phosphate

rock to estimate the P solubilizing capacity of two SOB strains Acidithiobacillus

ferrooxidans and Acidithiobacillus thiooxidans. They reported that both strains

together can solubilize huge quantity of P (70.2 % P2O5). They also determined

high quantity (39.81 mM) of sulfuric acid in the medium inoculated with

Acidithiobacillus thiooxidans and less quantity (28.83 mM) of sulfuric acid in the

medium inoculated with Acidithiobacillus ferrooxidans.

Rate of sulfuric acid produced directly relates to the rate of P dissolved.

Phosphorous solubilzation is a pH dependant phenomenon. Soil pH declines due

to H+ production as a result of S oxidation by SOB in soils (Jaggi et al., 2005;

Yang et al., 2010). Application of S may improve essential micronutrients

availability in soils having high pH values (Haneklaus et al., 2005). An increase in

electric conductivity and decrease in CaCO3 contents in the soil is also observed as

a result of H+ and SO42- production by S oxidation (Oh et al., 2010; Yang et al.,

2010). Furthermore, the concentration of other nutrients such as P and Zn also

20

increases in the soil solution by S oxidation which contributes to the EC values

(Zhou et al., 2002; Jaggi et al., 2005)

Liu et al. (2004) measured different quantities of sulfuric acid produced as

a result of biological S oxidation by Thiobacillus thiooxidans using different doses

of S in growth media. Maochun et al. (2002) and Kumar and Nagendran (2008)

recorded sulfuric acid production during S oxidation by Acidithiobacillus

thiooxidans (strain HSS). They also reported P solubilization (24 to 100 %) from

fluorapatite mineral present in phosphate rock. Stamford et al. (2003) and

Stamford et al. (2007) reported that Acidithiobacillus species produced sulfuric

acid by S application in soil which enhanced soil bioavailable P. Water-soluble

phosphorus level is increased by T. thiooxidans when compared with non-

inoculated treatments. Application of S and vermicompost also shows positive

effect on water soluble P (Aria et al., 2010) and the highest amount of water

soluble P was recorded in the first 15 days of incubation.

Miransari et al. (2007) reported that Thiobacilli enhanced biochemical S

oxidation through which soil pH decreased and P availability increased. According

to Besharati et al. (2007) application of S along with Thiobacillus significantly

increases plant available P in soil. Acidithiobacillus ferrooxidans and A.

thiooxidans oxidize pyrite and S, respectively and release P from RP (Rajan, 2002;

Besharati et al., 2007; Chi et al., 2007). Phosphorous solubilizing efficiency of

SOB can also be evaluated through determining phosphorous solubilizing index

21

(PSI) like other phosphorous solubilzing microorganisms (Hariprasad and

Niranjana, 2009; Ahmad and Khan, 2010).

Brenner et al. (2005) characterized and identified different SOB isolates

using different morphological, physiological and biochemical properties according

to the Bergey's Manual of Systematic Bacteriology. Similarly, various scientists

identified the genus Thiobacillus on the basis of their different biochemical

characteristics (Kelly and Harrison, 1989; Kelly and Wood, 2000; Vidyalakshmi

and Sridar, 2007; Kumar and Nagendran, 2008; Jiang et al., 2009; Hassan et al.,

2010; Babana et al., 2011).

From all the above mentioned discussion, it is concluded that mostly

calcium bounded insoluble and unavailable forms of P exist in alkaline and

calcareous soils of Pakistan. These fixed forms of P in soil can be released /

solubilzed by their reaction with sulfuric acid produced through bacterial S

oxidation by interactive effect of Thiobacillus spp. and S application in soil.

Availability of sulfates and soil micronutrients (Zn, Fe, Mn, B and Cu) is also

restricted by soil alkalinity and calcareousness and their bioavailability can be

enhanced through bacterial S oxidation mechanism. Therefore, exploitation of

Thiobacillus spp. for enhancing bio-available P in soil through bio-fertilization has

a great potential for making use of ever increasing fixed P pool in soil and natural

reserves of rock phosphate.

22

Chapter 3

MATERIALS AND METHODS

The materials and methods included sampling for sulfur oxidizing bacteria

from different microbial ecologies, isolation of efficient SOB, testing of

phosphorous solubilization potential of these SOB and comparison of different

sulfur levels with SOB against P fertilizer for P solubilization. Details of all the

experiments are described in the following paragraphs:

3.1 SCREENING OF SULFUR OXIDIZING BACTERIA FROM

DIFFERENT MICROBIAL ECOLOGIES

Samples were collected from ten different ecologies viz., paddy fields

(PF), wheat rhizosphere (WR), sugarcane rhizosphere (SR), maize rhizosphere

(MR), industrial wastewater (IW), canal water (CW), sulfur mud (SM), sewage

water (SW), industrial waste sludge (IS) and sewage sludge (SS). Detail of

sampling sites (ecologies) is given in Appendix I.

3.1.1 Isolation of Sulfur Oxidizing Bacteria

Isolation of SOB was carried out by using thiosulphate broth medium

(Beijerinck, 1904). Its composition is: Na2S2O3, 5.0 g; K2HPO4, 0.1 g; NaHCO3,

0.2 g; NH4Cl, 0.1 g dissolved in 1.0 L distilled water. The pH of the medium was

adjusted at 8.0. The indicator used was bromo cresol purple. The medium was

then autoclaved for sterilization. From the collected samples, 1 g mL-1 was added

22

23

to 20 mL of the broth poured in test tubes under aseptic conditions (Wollum II,

1982). Then the tubes were incubated at 30 °C in BOD incubator for 4-5 days.

Change in colour from purple to yellow indicated the growth of SOB in the tubes.

Purification of isolates was undertaken by transferring the isolates to the

fresh broth medium thrice at fortnightly intervals. Individual colonies were

obtained by streaking isolates on thiosulphate agar plates. Fifty pure isolates

obtained were labeled according to their sampling ecologies. The detail is given in

Table 1. These pure isolates were preserved for their characterization and further

experiments. Pure isolates of SOB the most efficient SOB were screened on the

basis of their (i) efficiency of lowering pH in thiosulphate broth medium (ii)

growth rate in thiosulphate agar plates (iii) phosphorous solubilization index (PSI)

and (iv) P solubilization efficiency. The screened SOB isolates were characterized

by colony morphology and biochemical tests (Smibert and Krieg, 1994). Detail of

procedures is as under:

3.1.2 Screening of Sulfur Oxidizing Bacteria for Phosphorous Solubilization

For pH reduction test thiosulphate broth medium was prepared and its pH

was adjusted at 8.0. One milliliter specimens of previously obtained isolates were

inoculated in 20 mL thiosulphate broth medium, and incubated at 30 °C for 16

days. The pH was recorded after 1, 2, 4, 8 and 16 days of incubation. Screening of

isolates was done on the basis of their efficacy to reduce pH from 8.0 to less than

6.0. Also colour change from purple to yellow in broth medium was noted.

24

For colour change test in thiosulphate agar plates, Thiosulphate agar plates

were prepared and isolates were inoculated. Rate of colour change (from purple to

yellow) was recorded after 01, 02, 04 and 08 days of inoculation to determine the

growth rate efficiency of isolates.

Phosphorous solubilization index (PSI) was determined by placing the

SOB culture (0.1 mL) on thiosulphate tricalcium phosphate (TCP) 0.5 % agar

plates and incubated for 8 days at 30 °C. Phosphorous solubilization zones were

formed on thiosulphate TCP agar plates. Phosphorous solubilization index was

calculated after 01, 02, 04 and 08 days using the following formula (Edi-Premono

et al., 1996).

Colony diameter + Halozone diameter

PSI = _______________________________

Colony diameter

Phosphorous solubilzation efficiency of SOB isolates was determined

through the following two bioleaching tests:

A test was performed to measure the phosphorous solubilization efficiency

of the isolates selected through pH, colour change and PSI measurements. The

experiment was arranged in completely randomized design with three replications.

Thirty three conical flasks of 250 mL were used which contained 100 mL

thiosulphate broth media in which K2HPO4 was replaced by tricalcium phosphate

(TCP 0.5 %). The pH was adjusted at 8.0. After autoclave the flasks were

25

inoculated with 1 mL broth culture of each of the 10 selected SOB isolates in 3

flasks and three flasks were kept as control without inoculation. The flasks were

incubated (100 rev min−1) at 30 °C for 32 days. At various intervals, aliquot

samples (5 mL) were drawn and centrifuged. The supernatants were examined for

pH, sulfate contents and P solubilization.

Another test was performed to test the P solubilization efficiency of the

best 07 SOB isolates among from the 10 isolates tested with TCP. The experiment

was organized in a completely randomized design (CRD) with three replications.

Twenty four conical flasks of 250 mL (including 03 as control) were used which

contained 100 mL thiosulphate broth media and 1 g elemental sulfur. The pH was

adjusted at 8.0. After autoclave the flasks were inoculated with 1 mL broth culture

of each of the seven selected SOB isolates in three flasks making three replications

and three flasks were kept as control without inoculation. The flasks were

incubated at 30 °C. Sulfuric acid produced thus was utilized in phosphorous

solubilization from the pre sterilized rock phosphate (10 g) added after 07 days of

inoculation. At various intervals aliquot samples (5 mL) were drawn. The

Supernatants were tested for pH, sulphate contents and for soluble P contents.

Metrohm High-precision 780 pH meter was used to determine pH of the

leach solutions and the amount of soluble P was determined through Mo-blue

method (Watanabe and Olsen, 1965). Sulfates concentration in the leach solutions

26

was measured by ion chromatography (conductivity detector L-2470, pump L-

2130, column oven L-2350) as described by Oh et al. (2010).

3.1.3 Biochemical Characterization of Selected Isolates

For Gram staining thin smears of the SOB isolates were made on the glass

slides and were heat fixed. Smears were stained with crystal violet. Slides were

flooded with iodine solution for 30 seconds. Iodine solution was drained and 75 %

alcohol was used for half to 1 minute for colour removal. Counter staining was

done with safranin for 1 minute. Then the glass slides were examined for colour

indication of G +ve and G -ve nature of bacteria (Vincent, 1970).

Motility test was performed for observing the ability of the SOB to move

away from the line of inoculation. Motility media (Barrow and Feltham, 1993)

was used for this purpose. Motility agar tubes were prepared by taking motility

agar 3.0 g/150 mL distilled water in 250 mL conical flasks. After autoclave it was

poured in glass tubes. To perform the motility test SOB samples were inoculated

in the motility agar tubes with a straight wire up to the depth of about 5 mm. After

capping the tubes, they were placed in the incubator at 30 °C for 48 hours. The

SOB which migrated away from the line of inoculation showed positive motility

test while the others indicated negative motility test result.

For catalase test a drop of hydrogen peroxide was kept on a microscope

slide. Colonies of the SOB were touched with an applicator stick and then pasted a

27

small in the drop of hydrogen peroxide. Formation of bubbles indicated catalase

positive test, whereas catalase test was negative in case of no bubbles formed.

For oxidase test prepared 1 % solution of N-tetramethyl-p-phenylen

ediamine dihdrochloride in distilled water. Then poured it in a filter paper. After

that took colonies of SOB with an applicator stick and pasted on the filter paper.

Colour change to purple within 30-60 seconds showed positive, while no colour

indicates oxidase test negative.

Nitrate reduction test was performed by taking Nitrate broth @ 9 g L-1

distilled water, dispensed 10 mL of broth into glass tubes and autoclaved them.

Glass tubes were heavily inoculated (1.0 mL/tube) with fresh cultures of SOB.

Also took a negative control along with others. Glass tubes were incubated at 35 to

37 °C for 48 hours. Then put 5 drops of reagent A first and then 5 drops of reagent

B into the test tubes. Glass tubes were shaked well. Red/pink colour developed

within a few minutes indicated nitrate reduction test positive. In case of colourless

suspension small amount of zinc powder was added. Colourless suspension for 10-

15 minutes revealed positive test. But in case of pink colour formation the result

was negative (Knapp and Clark, 1984).

Reagent A (sulfanilic acid solution): Dissolved sulfanilic acid @ 8 g L-1

5N acetic acid and stored it in room temperature in dark brown glass bottles.

Reagent B (α-Naphthylamine solution): Dissolved N, N-Dimethyl-1-

28

naphthylamine @ 6 g L-1 5N acetic acid and stored it at 2 to 8°C in dark brown

glass bottles.

For triple sugar test (glucose, sucrose, lactose and H2S production)

dissolved triple sugar iron (TSI) agar @ 65 g L-1 distilled water. Autoclaved it and

poured in already autoclaved slants. After cooling, the slants were inoculated with

SOB and placed in the incubator at 30 °C for 48 hours. Change in colour indicated

different positive and negative results as stated in the followings:

Red colour No fermentation of glucose, sucrose and lactose and

no H2S is formed

Yellow slant, yellow butt glucose, sucrose and lactose fermented

Black colour H2S formed

Red slant with yellow butt No lactose or sucrose fermentation, lactose is fermented

Methyl red (M.R.) test was carried out by dissolving methyl red-Vogues

Proskauer agar @ 15.0 g L-1 distilled water. After autoclave it was transferred into

already autoclaved slants. Then inoculated them with SOB to be tested with a

transfer loop and allowed to grow at 30 °C for 5 days in the incubator. After this,

methyl red was added in the MR tubes. Red colour indicated positive result and

yellow colour was due to negative response.

For Citrate test dissolved Simmons citrate agar @ 23.04 g L-1 distilled

water and autoclaved. Transferred it into already autoclaved slants. Then

29

inoculated them with SOB to be tested with a transfer loop and allowed to grow at

30 °C for 48 hours in the incubator. Colour changed from green to blue indicated

positive results.

3.2 COMPARATIVE EFFICIENCY OF THE ISOLATES FOR SOIL

PHOSPHOROUS SOLUBILIZATION

One kg soil was incubated and twenty treatments combinations were

applied with three replications. Two factor treatments viz., (i) three levels of S and

(ii) four strains of the most efficient Thiobacillus spp. (1 mL of 106 cells fresh

culture g-1 S) for P solubilization were employed. Facotor 1 consisted of S levels

(i) no S (ii) 25 mg kg-1 (iii) 37.5 mg kg-1 and (iv) 50 mg kg-1. Factor 2 contained

(i) control (No Thiobacilli spp. inoculation) (ii) Thiobacillus isolate IW16 (iii)

Thiobacillus isolate SW2 (iv) Thiobacillus isolate IW1 and (v) Thiobacillus isolate

IW14 strain. The soil was incubated at 30 °C for 90 days, and samples were drawn

after 30, 60 and 90 days. The samples were analysed for pH, ECe, CaCO3

contents, sequential P fractions, bio-available P, different sulfur fractions and

micronutrients concentration in the incubated soil.

3.3 SULFUR OXIDIZING BACTERIA APPLICATION ON PLANT

GROWTH AND PHOSPHOROUS UPTAKE

Effect of Thiobacillus strains (IW16 and SW2 @ 1 mL of 106 cells fresh

culture g-1 S) and S (100 kg ha-1) in combination with two doses of phosphorous

fertilizer viz., 45 and 90 kg P2O5 ha-1 on different attributes of soil and crops was

30

determined through field experiment. The experiment was conducted in two

permanent lay outs on two places by using two cereal based multiple cropping

systems, viz., rice- wheat on one place and maize-maize (spring and autumn) on

the other place. The plot size was 3 m × 3 m where fifteen treatment combinations

were arranged in randomized complete block design in three replications.

Recommended cultural and agronomic practices were followed equally in all

treatments. The treatments combinations were applied at the start of the

experiment. The treatments were (i) control P0S0, (ii) P1 45 kg P2O5 ha-1, (iii) P2

90 kg P2O5 ha-1, (iv) S 100 kg ha-1 and (v) P1S 45 kg P2O5 ha-1 + 100 kg S ha-1

taken as Factor 1, while factor 2 contained (i) control (No Thiobacilli spp.

inoculation), (ii) Thiobacillus strain1 (IW16 strain) and (iii) Thiobacillus strain2

(SW2 strain).

Rice nursery (C.V. Pukhraj) was transplanted on 1st July. Fifty five kg ha-1

N, full dose of potassium (K2O @ 70 kg ha-1) and two P doses in the form of DAP

(P1 = 45 kg P2O5 ha-1, and P2 = 90 kg P2O5 ha-1) were used as a basal dose.

Remaining half dose of nitrogen was applied after 25 days of rice nursery

transplantation. Soil samples were taken before rice nursery transplantation and at

harvest to analyze for quantity of bio-available P in the soil. Various crop

parameters (plant height, number of tillers plant-1, panicle length and number of

grains panicle-1) were recorded at harvest. Then the concentration of P in rice

grain and straw was determined. Phosphorous uptake by grains and straw was also

31

calculated. Rice paddy, straw and biological yields, harvest index and 1000 grains

weight were recorded by harvesting from the whole treatment plots.

Wheat (Triticum aestivum L) crop variety Millat 2011 was sown after the

harvest of rice crop on 1st November. The seed (125 kg ha-1) treated with fungicide

Vitavex @ 2.5 g kg-1 of seed were applied in combination at the start of the

experiment. Full recommended doses of P in the form of DAP (P1 = 45 kg P2O5

ha-1 and P2 = 90 kg P2O5 ha-1), potassium (K2O @ 70 kg ha-1) in the form of

potassium sulphate and half dose of nitrogen (55 kg ha-1) in the form of urea were

applied at sowing, while remaining half dose of nitrogen was applied at first

irrigation. Soil samples were drawn at harvest for determining the amount of bio-

available P. Plant sampling was done at harvest to record plant height, number of

tillers plant-1, spike length and number of grains spike-1. Then the plants were oven

dried (at 70 °C for 48 h) to determine P concentration in wheat grain and straw

and then P uptake by wheat grain and straw was also calculated. Whole of the

treatment plots were harvested and air dried to measure wheat grain straw and

biological yields, harvest index and 1000 grains weight.

Maize (Zea maize L) crop variety hybrid 32 M 15 was sown on 1st

February (spring crop) and 1st July (autumn crop) with seed rate of 30 kg ha-1. All

treatments were applied in combination at the start of the experiment. Nitrogen

fertilizer was divided into four equal doses and each dose contained 46 kg N ha-1.

32

First nitrogen dose as urea, full dose of potassium (K2O) @ 70 kg ha-1 as

potassium sulphate and phosphorous doses P1 (45 kg P2O5 ha-1) and P2 (90 kg P2O5

ha-1) as DAP were applied at the time of sowing. Second nitrogen dose was

applied at the time when the plants attained 1.0 feet height, third dose was given

when the plants reached the height of 2.5 feet, while fourth and the last dose of

nitrogen was applied to the crop just before the flowering stage. Soil sampling was

undertaken before and after harvest to determine bio-available P contents in the

soil. At harvesting stage plant sampling (10 plants per plot) was done for counting

the number of cobs plant-1 and number of grains plant-1. Plants samples were oven

dried at 70 °C to determine P concentration in maize grains and stalk. Crop from

whole of the treatment plots was harvested, sun dried and then grain, stalk and

biological yields were recorded and harvest indexes were determined.

Thousand grains weight in all the crops was recorded by counting

randomly selected 1000 grains (air dried) from each treatment. Similarly, P in all

the crops was calculated by the following formula:

P uptake (kg P2O5 ha-1) = P (concentration %) × Yield (kg) / 100

3.4 PLANT ANALYSES

For the determination of P concentration from straw / stalk and grains of

rice, wheat and maize crops di-acid (HNO3-HClO4) digestion was used (Ryan et

al., 2001). Took 1.0 g of already dried plant material samples and transferred them

separately into a 100-mL Pyrex digestion tubes. Ten mL of 2:1 nitric-perchloric

33

acid mixture was added and allowed to stand overnight. One tube was taken as

blank which contained 10 mL of 2:1 nitric-perchloric acid mixture without plant

material. Tubes were kept in a block digester and digestion was done. After

cooling the volume was made with distilled water.

Ammonium-vanadomolybdate reagent was prepared by dissolving

ammonium heptamolybdate (22.5 g) + ammonium metavanadate (1.25 g) +

concentrated nitric acid (250 mL) in 1.0 L distilled water. Took 10 mL of the

digested filtrate and 10 mL ammonium-vanadomolybdate reagent into 100-mL

volumetric flask and made the volume with distilled water. A calibration curve

was prepared by proceeding standard stock solution as for samples. A blank was

also made with 10 mL ammonium-vanadomolybdate reagent and proceeded as for

samples. The absorbance was read after 30 minutes at 410 nm wavelength in

spectrophotometer (Optizen 2120 UV plus) and P concentration was read from the

calibration curve.

3.5 SOIL SAMPLING AND ANALYSES

Soil samples were taken from 0-10 cm depth with 5 cm diameter auger.

Soil samples were sieved (2 mm) and stored for analyses (Ryan et al., 2001).

For determination of soil texture, suspension containing 40.0 g of soil

sample, 40 mL of 1% sodium hexa meta- phosphate and 150 mL of distilled water

was kept over night. Contents were shifted to cylinder after 10 minutes stir and

34

reading was recorded with Boyoucos Hydrometer method. Soil textural class was

checked through ISSS triangle (Page et al., 1982).

Soil pH was measured in 1:1 ratio of soil and distilled water by using soil

pH meter Metrohm High-precision 780 (Page et al., 1982). Soil extract was taken

from the saturated soil paste and determined electrical conductivity through

conductivity meter (Page et al., 1982).

Calcium carbonate was determined by a procedure based on CH3COOH

consumption. Known excess quantity of 0.4 M CH3COOH was added to a known

quantity of soil and pH of supernatant was measured after complete dissolution of

the solid phase carbonate (Leoppert et al., 1984).

For the determination of organic matter 1.0 gram sieved soil was taken,

added 10 ml 1 N potassium dichromate solution and 20 mL concentrated H2SO4.

After 30 minutes added 200 mL distilled water and 10 mL concentrated

orthophosphoric acid along with 10-15 drops of diphenylamine indicator. Titration

was done with 0.5 M ferrous ammonium sulfate solution (Page et al., 1982).

Available P of the soil samples was determined by Olsen et al. (1954)

method modified later (Watanabe and Olsen, 1965). Reagents used were sodium

hydroxide solution (5 N), sodium bicarbonate solution (0.5 M), sulfuric acid

solution (5 N), p-nitrophenol Indicator, 0.25 % w/v, reagent A (ammonium

35

heptamolybdate 12 g in 250 mL distilled water + antimony potassium tartrate

0.2908 g in 100 mL distilled water. Both the reagents were added to 1-L 5 N

H2SO4 (148 mL concentrated H2SO4 per liter) in a 2-L volumetric flask and made

the volume with distilled water) and reagent B (L-Ascorbic acid (C6H8O6) 1.056 g

+ 200 mL of Reagent A). Five gram soil (2-mm) was taken in a 250-mL

Erlenmeyer flask, added 100 mL 0.5 M sodium bicarbonate solution and shaken

for 30 minutes. One flask having all chemicals without soil was included as blank.

The solution was filtered. Took 10 mL filtrate in a 50-mL volumetric flask,

acidified to pH 5.0 with 5 N H2SO4 by using P-nitrophenol indicator (change in

colour from yellow to colorless). Added 8 mL reagent B and made the volume to

50- mL with distilled water. Then absorbance of blank, standards and samples

were read after 10 minutes at 882 nm wavelength in spectrophotometer (Optizen

2120 UV plus) and P concentration was read from the calibration curve.

3.5.1 Phosphorous Fractionation

Sequential extraction of P was carried out according to the procedure given

by Jiang and Gu (1989) for the determination of various P fractions in soil. A brief

methodology is given below:

Step 1: Ca2-P

One gram sieved soil was taken into 100 mL centrifuge tube, added 50 mL

of 0.25 M NaHCO3, shaken for 1 hour (20-25oC), centrifuged at 3500 revolutions

per minute for 8 minutes. Put the upper cleaned solution into the triangular flask,

36

took 5 mL of this solution into 50 mL volumetric flask and diluted to about 25 mL

with distilled water. Added two drops of 2, 4- DNP indicator, adjusted the colour

of the solution to light yellow with H2SO4 and NH4OH. Added 5 mL of colour

reagent B (as used in available P determination) and the volume was made to 50

mL. After standing for half an hour P determination was done by

spectrophotometer (Optizen 2120 UV plus) at 700 nm wavelength.

Step 2: Ca8-P

The soil residue from step 1 was washed twice with 95 % alcohol and kept

for 4 hours after adding 50 mL 0.5 M NH4AC (pH 4.2). The mixture was shaken

for 1 hour, centrifuged and the supernatant was analyzed for P as for first step.

Step 3: Al-P

The soil residue was washed twice with saturated NaCl solution, shaken

for 1.0 hour after adding 50 mL 0.5 M NH4F (pH 8.2). Then the mixture was

centrifuged. Took 10 mL of the supernatant into 50 mL volumetric flask with

same volume of 0.8 M H3BO3, made the volume and was analyzed for P as for

first step.

Step 4: Fe-P

The soil residue was washed twice with saturated NaCl solution, shaken

for 2 hours after adding 50 mL solution of 0.1M NaOH + 0.1M Na2CO3 in 1:1

ratio. It was left unshaken for 16 hours, shaken again for 2 hours and centrifuged.

37

Put the upper cleaned solution into the triangular flask, added 1.5 mL of

condensed H2SO4 (The volume was included when determining P content), shaken

and filtered in order to wipe out flocculated organic matter. Took 5 mL of the

solution into 50 mL volumetric flask and measured P as for step one.

Step 5: Occluded-P

The soil residue was washed twice with saturated NaCl solution, kept in

hot water bath after adding 40 mL 0.3 M trisodium citrate solution. After attaining

80 °C temperature, 1 g of sodium dithionite was added and the suspension was

stirred continuously for 15 minutes. Then added 10 mL of 0.5N NaOH solution,

continued stirring for 10 minutes, centrifuged and cooled off. . Took 10 mL of the

filtrate into 50 mL triangular flask and added 10 mL of the three acid mixture

solution (1 H2SO4: 2 HClO4:7 HNO3). Made the volume after digestion and

analyzed P.

Step 6: Ca10-P

Fifty milliliter of 0.5 N H2SO4 was added to the remaining soil residue,

shaken for 1 hour and centrifuged. Took 5 mL of the filtrate into 50 mL

volumetric flask, made the volume and determined P as for step one. Quantity of P

in each fraction was determined by following the procedure of Murphy and Riley

(1962).

Total P was determined by digesting the soil with HClO4 method (Olsen

and Sommers, 1982). Two grams air-dried soil (0.15 mm) was taken in digestion

38

tube and 30 mL of 60 % HClO4 along with few pumice-boiling granules were

added. The tubes were placed in a block-digester; its temperature was raised to

180 °C till the white sand colour, then cooled, raised to 100 mL volume and

filtered through Whatman No. 41 filter paper. Ten mL vanadomolybdate reagent

was added into 5 mL of the sample filtrate making volume to 50 mL. Absorbance

of blank, standards and samples were read on spectrophotometer (Optizen 2120

UV plus) at 410 nm wavelength.

Sulfur fractionation was done by the method followed by Morche (2008).

Step 1: S-H2O

One gram fine air dried sieved soil (<2 mm) was taken in 100 mL

centrifuge tube, shaken for 30 minutes after adding 10 mL de-mineralized water,

centrifuged for 10 minutes and the supernatant was analyzed for water soluble S.

Step 2: S-NaH2PO4

NaH2PO4 0.032 M 10 mL was added into the soil residue, shaken for 30

minutes, centrifuged at 10000 rpm for 10 minutes and the supernatant was

analyzed for sorbed S.

Step 3: S-HCl

HCl 1M 20 mL was added into the remaining soil sample, shaken for 60

minutes, centrifuged and supernatant was analyzed for occluded S.

39

Amount of S in each fraction was analyzed by ion chromatography

(conductivity detector L-2470, pump L-2130, column oven L-2350) adopting the

method described by Oh et al., 2010.

Total soil S was determined by gravitation method (Mohammed and

Adamu, 2009). Took 1.0 gm sieved (0.2 mm) soil in a crucible, mixed with 5.0 g

Na2CO3 and 0.2 g of NaNO3, heated in an electric muffle furnace model LEF-130

SE (3) at 400°C for 30 minutes. Then it was fused at 950 °C. The crucible was

cooled and placed in a 150 mL beaker. The beaker was heated below boiling on a

hot plate after adding de-ionized water to disintegrate the contents thoroughly.

The contents were then filtered into a 100 mL volumetric flask along with

sufficient deionized water, added 20 mL of 6 M HCl and made the volume.

Slowly added 10 mL BaCl2 2H2O (10 %) into the solution at boiling for

precipitation of sulphate. After cooling, it was filtered and washed the residue

with de-ionized water. Ignition of ashless filter paper was done at 40°C and

weighed the precipitate. HF and H2SO4 (few drops) were used to treat the ignited

precipitate. It was weighed again after cautiously ignited. Sulfur percent in the

precipitate was calculated by Jackson (1958).

Soil micronutrients (Zn, Fe, Cu, and Mn) were determined using the

method given by Ryan et al. (2001). Standard curves for each metal element were

prepared separately from their respective standard stock and working solutions on

atomic absorption spectrophotometer model AA-6300. Took 10 g soil (2-mm) into

40

a 125-mL Erlenmeyer flask, added 20 mL DTPA extraction solution, shaken for

two hours and was filtered. A blank was also run along with samples without soil.

Soil micronutrients (Zn, Fe, Cu, and Mn) were determined from the filtrate on

atomic absorption spectrophotometer.

The concentration of available B in the soil was determined by hot-water

method presented by Ryan et al. (2001).

3.6 STATISTICAL ANALYSES

During the first phase of study, variance in pH change, rate of colour

change, PSI, sulfate contents and quantities of P solubilized were analyzed taking

SOB collected from different ecologies as source of variance. In the second phase

the data regarding different soil variables were examined for taking different

levels of S amendment and Thiobacillus spp. as source of variance. In the third

phase (field experiments) S, two levels of P and Thiobacillus spp. were kept as

source of variance to analyze the data regarding soil and crop variables. Treatment

means were compared by DMRT at 5 % level of significance using MSTAT-C

software (Steel et al., 1997). The data was analyzed for simple linear correlation

and regression through MS Excel to evaluate the interrelationship and

interdependence among various soil and plant variables.

41

Chapter 4

RESULTS AND DISCUSSION

4.1 SCREENING OF SULFUR OXIDIZING BACTERIA FROM

DIFFERENT MICROBIAL ECOLOGIES

4.1.1 Occurance of Sulfur Oxidizing Bacteria

Among all the samples (160), 50 samples were SOB +ve (Table 1). The

industrial wastewater, sulfur mud and sewerage water had 80, 60 and 60 % SOB

occurrence frequency (Figure 2). Sulfur or reduced S compounds are essential for

the existence of Thiobacilli (Pokorna et al., 2007). A close SOB-S relationship

exists in biological S oxidation (Briand et al., 1999).

4.1.2 Screening of Sulfur Oxidizing Bacteria for Phosphorous Solubilization

The SOB isolates were screened on the basis of pH reduction in

thiosulphate broth, rate of colour change (purple to strong yellow) in thiosulphate

agar plates, phosphorous solubilization index (PSI) and their P-solubilization

efficiency through bioleaching tests in broth containing tricalcium phosphate and

Rock phosphate. Detail of results is as under:

4.1.2.1 The pH reduction

Amongst from 50 SOB isolates, the maximum significant decrease

in pH was observed with IW16 where pH decreased to 2.42 from pH 8.00 of the

the media after 16 days and minimum decrease was noted in SM9 giving a pH

41

42

Table 1. Ecology-wise description of sulfur oxidizing bacteria

Ecology Total SOB +ve

SOB -ve

Isolates

--------- No. of samples --------

Paddy fields (PF) 15 2 13 PF2, PF3

Wheat Rhizosphere (WR)

50 10 40 WR2, WR4, WR7, WR9, WR10, WR12, WR13, WR14, WR15, WR16

Sugarcane Rhizosphere (SR)

15 2 13 SR2, SR8

Maize Rhizosphere (MR)

15 2 13 MR6, MR8

Industrial wastewater (IW)

10 8 2 IW1, IW3, IW4, IW5, IW7, IW13, IW14, IW16

Canal water (CW) 10 3 7 CW1, CW2, CW3

Sulfur mud (SM) 15 9 6 SM1, SM2, SM3, SM4, SM7, SM9, SM11, SM12, SM14

Sewage water (SW) 10 6 4 SW1, SW2, SW4, SW5, SW11, SW14

Industrial waste sludge (IS)

10 5 5 IS1, IS2, IS11, IS12, IS16

Sewage sludge (SS) 10 3 7 SS1, SS4, SS6

Total : 160 50 110

43

0

15

30

45

60

75

90

PF WR SR MR IW CW SM SW IS SS

SOB Ecologies

SO

B o

ccu

ran

ce f

req

uen

cy (

%)

Figure 2. Frequency of SOB in sampling ecologies indicating highest number

different SOB in industrial waste water: PF, paddy fields, WR, wheat rhizosphere,

SR, sugarcane rhizosphere, MR, maize rhizosphere, IW, industrial wastewater,

CW, canal water, SM, sulfur mud, SW, sewage water, IS, industrial waste sludge,

SS, sewage sludge

44

Plate 1. Isolation and purification of sulfur oxidizing bacteria

45

value of 7.06 after 16 days (Appendix II). Six SOB isolates IW1, SW2, IW13,

IW14, IW16 and SM1 decreased net pH > 4.00 points and 01 SOB isolate SS1

reduced net pH 3.54 points. Thirteen isolates dropped net pH in the range of 2.00

to 2.90 points, 28 isolates reduced net pH from 1.00 to 1.90 points and 02 isolates

dropped net pH < 1.00 point. However, no change in pH was observed in case of

control where no inoculation was done. The SOB isolates which dropped

maximum pH after 16 days of inoculation were considered the best amongst the

50 SOB isolates. Four isolates viz., IW16, SW2, IW1 and IW14 were the most

efficient isolates in reducing pH, SM1 and IW13 isolates had moderate and the

rest of SOB isolates had poor efficiency of reducing pH in thiosulphate broth

media. The pH reduction in the media is linked with the production of sulfuric

acid by SOB isolates through S oxidation. High sulfuric acid producer SOB

isolates dropped more pH than the less sulfuric acid producers. Hassan et al.

(2010) and Yang et al. (2010) also reported decrease in pH after inoculation of

SOB as a consequent of sulfuric acid production through S oxidation.

4.1.2.2 Colour change Appendix III presents the data regarding rate of colour change in

thiosulphate agar plates inoculated with SOB isolates. Rate of colour change from

purple to yellow on thiosulphate agar plates inoculated with the SOB isolates

relate with efficiency of SOB isolates (Vidyalakshmi and Sridar, 2007). Amongst

IW1, SW2, SS1, IW13, IW14, IW16 and SM1 started changing colour during the

1st day. Amongst these seven SOB isolates SW2 and IW16 changed 100 % colour

46

in two days and IW1, IW14 and SM1 changed the colour in 4 days and the rest

two isolates SS1 and IW13 changed the colour in 08 days. No colour change was

found where no inoculation was done (control). Kelly and Wood (2000) reported

colour change by SOB in the media as an indication of sulfuric acid production.

Similarly, these results are quite similar with the results reported by Vidyalakshmi

and Sridar (2007) who also tested the efficiency of SOB isolates on the basis of

colour change in thiosulphate agar plates and then characterized different SOB

isolates on basis of rate of colour change.

4.1.2.3 Phosphorous solubilization index

The highest PSI 9.83 recorded in case of the isolate IW16 followed by PSI

8.42 for SW2, while the lowest PSI 0.86 was noted in case of SW5 (Appendix IV).

No holozone in thiosulphate agar plates was observed for control.

The isolates IW1, SW2, SS1, IW13, IW14, IW16, and SM1 made

holozones during 01 day indicating sulfuric acid production immediately after

inoculation which solubilized TCP around them, and, consequently holozones

appeared. Twelve SOB isolates were slower in producing sulfuric acid and

holozones became visible during 2nd day. Amongst, IW1, IS1, IS2, SW1, CW3,

WR4, WR10, WR13, and SM11 non significant difference was found in the SOB

isolates. The isolates PF2, IW5, IS11, SW4, SW5, CW2, WR2 and MR8 were the

slowest and the least efficient in making holozones. Islam et al. (2007) checked P

solubility of different phosphorous solubilizing bacteria on the basis of PSI and

47

reported the range of PSI from 1.2 to 6.7. Similarly, Hariprasad and Niranjana

(2009) and Ahemad and Khan (2010) also adopted the same method for estimating

PSI to evaluate P solubilizing efficiencies of different bacterial strains.

Seven SOB isolates IW1, SW2, SS1, IW13, IW14, IW16 and SM1 which

initiated colour change in thiosulphate agar plates and started making holozones in

in day 1st were selected for the next phase. Similarly, three SOB isolates WR12,

SM3 and SW11 performed better in colour change and holozone formation from

the 2nd day were also selected. In this way total 10 SOB isolates were selected for

the next assessment phase.

4.1.2.4 Phosphorous solubilization efficiency

Selected isolates were tested for their P solubilization capability inoculated

in thiosulphate TCP 0.5 % broth media for 32 days and recorded pH, sulfate

contents and quantity of P solubilized after 08, 16, 24 and 32 days. In the second

test rock phosphate was used instead of TCP and pH, sulfate contents and

quantities of P solubilized were recorded after 10, 20, 30 and 40 days.

Through tricalcium phosphate bioleaching

The highest reduction in pH was recorded as 2.46 (net reduction of 5.54

points) in IW16, while the lowest decrease in pH (4.52 with net reduction of 3.48

points) was noted in treatment SM3 (Table 2). However, pH of the control flasks

where no inoculation was done remained unchanged during the whole incubation

period. Four SOB isolates IW16, SW2, IW1 and IW14 were the best in pH

48

Table 2. The pH reduction by sulfur oxidizing bacteria in tricalcium phosphate

media during the incubation period

Treatments 08-day 16-day 24-day 32-day

Control 8.00 a 8.00 a 8.00 a 8.00 a

IW1 3.40 i 3.23 h 2.84 h 2.63 i

SW2 3.31 j 3.12 hi 2.78 h 2.58 i

SS1 5.06 e 4.49 e 3.54 e 3.24 e

WR12 7.00 c 5.61 c 4.48 c 4.22 c

SM3 7.18 b 7.00 b 4.86 b 4.52 b

IW13 3.74 g 3.54 g 3.13 g 2.88 g

IW14 3.62 h 3.46 g 3.03 g 2.76 h

IW16 3.23 k 3.01 i 2.72 h 2.46 j

SM1 4.48 f 3.88 f 3.33 f 3.03 f

SW11 5.36 d 4.94 d 4.08 d 3.54 d

LSD 0.05 0.16 0.17 0.08

p ≥ F 0.05. Similar letter (s) values in a column are not statistically different.

49

Table 3. Sulfates production by sulfur oxidizing bacteria in tricalcium phosphate

media during incubation period


------------------------------- mg L-1---------------------------------

Control 0.0 i 0.0 j 0.0 k 0.0 k

IW1 381.5 c 561.2 c 1392.8 c 2233.9 c

SW2 469.7 b 721.5 b 1582.0 b 2552.3 b

SS1 8.4 g 30.8 g 276.2 g 562.0 g

WR12 0.0 i 2.4 i 31.9 i 57.6 i

SM3 0.0 i 0.0 j 13.4 j 29.3 j

IW13 172.8 e 275.7 e 705.9 e 1279.8 e

IW14 230.9 d 331.0 d 901.3 d 1676.2 d

IW16 571.6 a 938.3 a 1817.7 a 3315.9 a

SM1 31.6 f 124.2 f 453.4 f 899.0 f

SW11 4.2 h 11.0 h 79.7 h 274.8 h

LSD 0.2 0.5 8.4 11.2


50

reduction in thiosulphate tricalcium phosphate broth media as well as in

thiosulphate media. Aria et al. (2010) and Oh et al. (2010) also recorded pH

reduction by Thiobacilli in different media. They declared it as an essential

characteristic of the genus Thiobacillus.

Amongst the ten SOB isolates IW16 produced the highest significant

amount of sulfates (3315.9 mg L-1), while the lowest quantity of sulfates (29.3 mg

L-1) were recorded with SM3 isolate (Table 3). Moreover, four SOB isolates

IW16, SW2, IW1 and IW14 were highly efficient in sulfate production than the

rest of seven isolates. The sulfate contents gradually increased from 8 to 32 days

of leaching time in all treatments except in control where no change was observed.

The increasing concentration of SO42- in the leach solutions depicted the efficiency

of the isolates to oxidize S. The most efficient SOB isolates rapidly oxidized S

compounds and converted them into sulfates, whereas less efficient SOB

isolates did this slowly and consequently low quantity of sulfates were

present in their leach solutions. Therefore, SOB isolates could be scrutinized on

the basis of sulfate concentration detected from their leach solutions (Lee et al.,

2005 and Yang et al., 2010).

The isolates which have the highest potential to produce sulfuric acid

through bacterial S oxidation solubilized maximum quantity of P from tricalcium

phosphate, while the isolates possessing less efficiency to oxidize S compounds

dissolved minimum amount of P in the leaching media. Thiobacillus isolate IW16

51

Table 4. Phosphorous solubilization by sulfur oxidizing bacteria in tricalcium



-------------------------- mg L-1------------------------------------

Control 0.0 k 0.0 k 0.0 k 0.0 k

IW1 472.0 c 630.2 c 702.1 c 807.9 c

SW2 497.2 b 681.8 b 759.2 b 901.7 b

SS1 308.1 g 344.4 g 381.6 g 436.2 g

WR12 257.3 i 288.4 i 314.1 i 371.7 i

SM3 202.3 j 269.7 j 306.1 j 364.7 j

IW13 434.2 e 501.0 e 610.2 e 705.8 e

IW14 458.9 d 581.7 d 687.2 d 751.4 d

IW16 531.1 a 761.2 a 819.9 a 954.2 a

SM1 334.3 f 378.3 f 421.8 f 454.5 f

SW11 275.6 h 303.7 h 342.1 h 396.7 h

LSD 1.7 1.6 1.5 1.6


52

released 954.2 mg L-1 of P and remained the highest amongst the ten SOB

isolates, whereas the lowest performance was noted in case of SM3 that

solubilized 364.7 mg L-1 of P (Table 4). Soluble P contents were maximum in first

16 days in all treatments. It indicated that maximum S oxidation occurred during

the first 16 days. Aria et al. (2010) also recorded maximum P solubilization

through S oxidation by Thiobacilli in first 15 days after incubation. Similarly,

Kumar and Nagendran (2008) reported P solubilization by Acidithiobacillus

thiooxidans during a bioleaching experiment.

Data regarding pH change, sulfate contents, and quantity of P solubilized

revealed that the isolates WR12, SM3 and SW11 predicted very ordinary results

and hence, the remaining seven isolates were selected for further analytical phase.

Simple linear correlations among pH, sulfate contents and P solubilization

are presented in Table 5. Negative significant correlation existed between the

SO42- contents and P solubilized (Figure 3). Also negative correlation existed

between pH and P solubilized. It showed that the relationship was linear and

significant. The results conformed with Stamford et al. (2003) and Bhatti and

Yawar (2010) who also reported strong negative significant correlation between

pH and SO42- contents and huge positive significant association between sulfate

contents and the amount of P solubilized through bacterial S oxidation

mechanism.

53

Table 5. Correlation among various variables in tricalcium phosphate media

Correlation parameters pH Sulfates

After 08 days

SO42- -0.77**

P solubilized -0.95** 0.80**

After 16 days

SO42- -0.72*


After 24 days

SO42- -0.67*


After 32 days

SO42- -0.65*


* p ≥ F 0.05 ** p ≥ F 0.01

54

y = -86.388x + 769.71

R2 = 0.91

0

200

400

600

800

1000

2 3 4 5 6 7 8

pH of leach solution

P s

olub

ilize

d (m

g L-1

)

y = -121.72x + 987.29

R2 = 0.83

0

200

400

600

800

1000

2 3 4 5 6 7 8


P s

olub

ilize

d (m

g L-1

)y = -144.31x + 1047.2

R2 = 0.79

0

200

400

600

800

1000

2 3 4 5 6 7 8


P s

olub

ilize

d (m

g L-1

)

y = -154.61x + 1118.9

R2 = 0.73

0

200

400

600

800

1000

2 3 4 5 6 7 8


P s

olub

ilize

d (m

g L-1

)

Figure 3. Phosphorous solubilization relation with pH in tricalcium phosphate

media changes: a to d represent data from day 08, 16, 24 and 32, respectively

(d)

(a)

(c)

(b)

55

Through rock phosphate bioleaching

The isolates decreased pH significantly as compared with control after 07,

10, 20, 30 and 40 days of leaching period (Table 6). After 07 days the highest

significant decline in pH (1.44 with net decrease of 6.56 points) was recorded with

IW16 and the lowest pH decrease (2.53 with net decrease of 5.47 points) was

noted with SS1. Then rock phosphate was added into the flasks at this stage due to

which pH of the inoculated 07 flasks quickly increased by alkaline minerals

(calcite and lime) present in rock phosphate.

Three days after rock phosphate addition, minimum pH (3.48) was

observed in IW16 medium and maximum pH (7.10) was recorded in SS1 medium.

Then again the pH started decreasing due to sulfuric acid generation by SOB

isolates as a product of biological S oxidation. Continuous decrease in pH was

recorded in 20th to 40th day of incubation. Amongst the seven SOB isolates the

highest significant decrease in pH (2.58) was again noted in IW16 while the lowest

pH reduction (3.92) was recorded in SS1. No change in pH was noted in the

control flasks before / after rock phosphate addition. Results predicted that

treatments containing IW16, SW2, IW1 and IW14 performed efficiently in

reducing pH significantly. Bhatti and Yawar (2010) recorded almost similar

results of pH reduction by two SOB strains Acidithiobacillus ferrooxidans and

Acidithiobacillus thiooxidans in a rock phosphate bioleaching experiment.

Furthermore, Kelly and Wood (2000) and Stamford et al. (2003) reported pH

reduction by Thiobacilli through biological S oxidation.

56

Table 6. The pH reduction by sulfur oxidizing bacteria in rock phosphate leach

suspension

Treatments 07-day 10-day 20-day 30-day 40-day

Control 8.00 a 8.00 a 8.00 a 8.00 a 8.00 a

IW1 1.50 f 4.02 f 3.85 f 3.35 f 2.77 f

SW2 1.47 fg 3.69 g 3.58 g 3.00 g 2.68 g

SS1 2.53 b 7.10 b 7.00 b 5.36 b 3.92 b

IW13 1.77 d 5.11 d 4.81 d 4.00 d 3.02 d

IW14 1.62 e 4.36 e 4.12 e 3.64 e 2.89 e

IW16 1.44 g 3.48 h 3.33 h 2.88 h 2.58 h

SM1 1.93 c 5.39 c 5.04 c 4.25 c 3.40 c

LSD 0.06 0.11 0.10 0.08 0.08


57

Table 7. Sulfates production by sulfur oxidizing bacteria in rock phosphate leach

suspension


-------------------------------- mg L-1-------------------------------------

Control 0.0 h 0.0 g 0.0 g 0.0 h 0.0 h

IW1 30155.8 c 90.6 c 135.8 c 427.5 c 1606.5 c

SW2 32541.4 b 194.3 b 251.3 b 965.0 b 2115.5 b

SS1 2825.0 g 0.0 g 0.0 g 4.2 g 115.0 g

IW13 16124.5 e 7.4 e 15.0 e 95.7 e 909.7 e

IW14 22760.3 d 41.8 d 72.2 d 220.0 d 1227.2 d

IW16 34766.9 a 317.8 a 453.3 a 1262.0 a 2333.8 a

SM1 11159.9 f 3.9 f 8.6 f 54.2 f 384.5 f

LSD 47.0 1.3 2.3 3.9 1.5


58

Sulfates produced by the isolates during biological S oxidation are

presented in Table 7. Maximum sulfates concentration was recorded after 07 days

of incubation in all the isolates compared to control. Sulfates concentration

decreased with the lowest sulfate was noted after 10 days of incubation. Reason

was the consumption of excess sulfates in reaction with alkaline minerals (calcite

and lime) present in rock phosphate to form precipitation as CaSO4 (Bhatti et al.,

2010). Then again increased quantity of sulfates was observed from 20th to 40th

days of incubation. Maximum significant amount of sulfates (2333.8 mg L-1) was

produced by IW16 and the minimum quantity was recorded (115.0 mg L-1) with

SS1. Control flasks had no SO42- through out the incubation period. Sulfates

production by SOB and especially by the genus Thiobacilli as an end product of

bacterial S oxidation were also reported by Ohba and Owa (2005), Kumar and

Nagendran (2008) and Yang et al. (2008).

Phosphorous solubilization from rock phosphate under the influence of the

isolates is presented in Table 8. Soluble P increased from 10th day to the 40th day

in all treatments except in control where no change in P contents was recorded

during the whole period. The highest soluble P (7987.9 mg L-1) was recorded with

the isolate IW16 and the lowest soluble P (2391.9 mg L-1) was recorded with SS1.

Rock phosphate contained calcite (CaCO3) and lime (CaO) in large

quantities, therefore, these two gangue minerals consumed major portion of

59

Table 8. Phosphorous solubilization by sulfur oxidizing bacteria in rock phosphate

media


----------------------------------- mg L-1 ----------------------------------

Control - 0.7 h 0.7 h 0.7 h 0.7 h

IW1 - 623.8 c 1753.7 c 2564.1 c 6757.0 c

SW2 - 684.6 b 1985.5 b 3086.3 b 7582.1 b

SS1 - 49.8 g 543.2 g 1282.6 g 2391.9 g

IW13 - 392.0 e 1187.3 e 1977.5 e 4368.2 e

IW14 - 510.6 d 1576.3 d 2187.8 d 5860.0 d

IW16 - 745.9 a 2219.8 a 3278.5 a 7987.9 a

SM1 - 248.0 f 954.7 f 1634.5 f 3602.4 f

LSD 1.7 2.4 35.1 52.0


60

sulfuric acid and consequently gypsum (CaSO4∙2H2O) was formed. Bhatti et al.

(2010) also confirmed gypsum formation as a result of chemical reaction between

sulfuric acid and the alkaline minerals. Mechanism of P solubilization and gypsum

formation from rock phosphate was also reported by Bhatti and Yawar (2010).

Sulfuric acid and phosphoric acid (formed during the process) attacked on

fluorapatite and released P in the form of plant available dicalcium P.

With the passage of time pH in the growth media dropped due to sufficient

amount of biologically generated sulfuric acid and high concentration of soluble P

was recorded. Fast release of P from rock phosphate was noted at low pH values.

The extent of P solubilization had positive significant correlation with the amount

of bacterially generated sulfuric acid in bioleaching process. Greater concentration

of sulfuric acid resulted in high P solubilization rate and low quantity of sulfuric

acid caused low P solubilization in the leach solutions. Therefore, the importance

of sulfuric acid in P dissolution from rock phosphate was well established.

High sulfuric acid producing SOB isolates were more efficient in P

solubilization than low sulfuric acid producing SOB isolates. The Isolates W16,

SW2, IW1 and IW14 were more efficient SOB isolates than IW13, SM1 and SS1

in P solubilization from rock phosphate. Maochun et al. (2002) reported P

dissolution by SOB from 24 to 100 % in an earlier study. Similarly, Bhatti and

Yawar (2010) recorded P solubilization from rock phosphate by Thiobacilli in the

range of 41.8 to 70.2 %.

61

Table 9. Correlation among various variables in rock phosphate media

Correlation parameters pH Sulfates

After 10 days

SO42- -0.71*

P solubilized -0.98** 0.80*

After 20 days

SO42- -0.68

P solubilized -0.97** 0.80*

After 30 days

SO42- -0.64


After 40 days

SO42- -0.67


* p ≥ F 0.05 ** p ≥ F 0.01

62

y = -0.1696x + 1.2795

R2 = 0.95

0

2

4

6

8

10

2 3 4 5 6 7 8

pH of leach suspension

P so

lubi

lize

d (g

L-1)

y = -0.4357x + 3.4415

R2 = 0.95

0

2

4

6

8

10

2 3 4 5 6 7 8


P so

lubi

lize

d (g

L-1)

y = -0.6025x + 4.5984

R2 = 0.93

0

2

4

6

8

10

2 3 4 5 6 7 8


P so

lubi

lize

d (m

g L-1

)

y = -1.2928x + 9.5475

R2 = 0.72

0

2

4

6

8

10

2 3 4 5 6 7 8


P so

lubi

lize

d (g

L-1)

Figure 4. Relation of P solubilization with pH in tricalcium phosphate leach

suspension changes: a to d represent data from day 10, 20, 30 and 40, respectively

(d)

(a)

(c)

(b)

63

The pH had negative significant correlation with SO42- contents and

quantity of P solubilized. While SO42- contents were positively and significantly

correlated with the concentration of P dissolved (Table 9). The pH and the

concentration of P solubilized had a linear relationship (Figure 4). Besharati et al.

(2007) and Miransari et al. (2007) also reported similar conclusions and found a

negative extensive association between pH and amount of P solubilized as a

consequent of bacterially produced sulfates and sulfuric acid by Thiobacilli.

4.1.3 Characterization and Identification of Sulfur Oxidizing Bacteria

Morphological, physiological and biochemical characteristics of the

selected 07 isolates are given in Table 10. All the isolates were found gram

negative and short rods but they showed difference in the utilization of S sources.

The isolates IW1, SW2, IW14 and IW16 utilized both elemental S and

thiosulphate and the isolates SS1, IW13 and SM1 utilized Thiosulphate only.

Three isolates IW1, SW2 and IW16 had smooth round and yellow coloured

colonies, isolates SS1 and IW13 had smooth round and pink coloured colonies,

while the isolates IW14 and SM1 had smooth, round and white coloured colonies.

The isolates IW16, SW2 and IW1 were high sulfate producing, acidophilic and pH

reducing isolates, the isolates IW14 and IW13 were high sulfate producing and pH

reducing, isolates, the isolates SM1 and SS1 were found moderate sulfate

producing and pH reducing. The isolates IW1, SW2, IW14 and IW16 were

autotrophic and as they did not utilize glucose whereas SS1, IW13 and SM1

utilized glucose and were declared as heterotrophic. Biotin effect was observed

64

Table 10. Biochemical characterization of the isolates

Characteristics IW1 SW2 SS1 IW13 IW14 IW16 SM1

Morphology SR SR SR SR SR SR SR

Gram reaction - - - - - - -

Elemental S° utilization + + - - + + -

Thiosulphate utilization + + + + + + +

Colony character SRY SRY SRP SRP SRW SRY SRW

pH reduction +++ +++ + ++ ++ +++ +

Sulfates production +++ +++ + ++ ++ +++ +

Nutritional type AT AT HT HT AT AT HT

Boitin Effect + + + + + + +

Motility M M NM M M M NM

Catalase - + - + - - -

Oxidase + + + + + + +

Nitrate reduction + + - + + + +

Glucose - - + + - - -

Sucrose - - - - - - -

Methyl red - - - - - - -

Citrate - + - - + - -

Carbohydrate hydrolysis

- - - - - - -

Indole Acetic acid (mg L-1)

15.25 32.65 - - 14.34 39.50 -

SR: short rod, SRY: smooth, round, yellow: SRP: smooth, round, pink: SRW: smooth, round, white, AT: Autotrophic, HT: Heterotrophic, M: motile, NM: non motile

65

positive for all the seven isolates. In motility agar medium, the isolates IW1, SW2,

IW13, IW14 and IW16 were observed motile, while SS1 and SM1 were found

nonmotile. Catalase test was positive for SW2 and IW13 isolates and it was

negative for IW1, SS1, IW14, IW16 and SM1 isolates. Oxidase test for all the

seven isolates was positive. Nitrate reduction test was found positive for IW1,

SW2, IW13, IW14 and IW16 and negative for SS1 and SM1 SOB. All the isolates

had no H2S production tested for H2S production. Similarly, sucrose and MR tests

were negative for all the isolates. Two isolates SW2 and IW14 had citrate test

positive, and the isolates IW1, SS1, IW13, IW16 and SM1 were negative.

Carbohydrate test was also negative for all the seven isolates. The SOB isolates

IW16, SW2 and IW1 and IW14 were indole acetic acid producers and they

generated 39.50, 32.65, 15.25 and 14.34 mg L-1 of indole acetic acid, respectively.

According to Bergey's Manual of Systematic Bacteriology (Brenner et al.,

2005), all the seven selected SOB isolates were gram negative and short rods. The

selected SOB isolates were recognized as Thiobacillus spp. because of their ability

to utilize S or thiosulphate as the only source of energy and carbon dioxide as a

sole source of carbon. Furthermore, they predicted high efficiency to produce

sulfates and reduced pH of the growth media. Therefore, these seven SOB isolates

belonged to the genus Thiobacillus. Several scientists adopted similar procedure

for the identification of Thiobacillus spp. (Kelly and Harrison, 1989; Kelly and

Wood, 2000; Kantachote and Innuwat, 2004; Lee et al., 2005; Vidyalakshmi and

Sridar, 2006-2007; Yang et al., 2008; Babana et al., 2011).

66

4.2 COMPARATIVE EFFICIENCY OF THE ISOLATES FOR SOIL

PHOSPHOROUS SOLUBILIZATION

4.2.1 Basic Soil Analyses before Incubation

The soil used in the incubation study was nonsaline, alkaline and

calcareous (Table 11). Total and plant available P in the soil was 664.7 mg kg-1

and 4.5 mg kg-1, respectively. Plant available P was low and came under deficient

category. Ahmad et al. (2006) reported total soil P in the range 652 to 1245 mg kg-

1. Pakistani soils were mostly P deficient as reported by Rehman et al. (2000) and

Solangi et al. (2006). The experimental soil contained total S 214.5 mg kg-1 and

water soluble sulfate was 9.6 mg kg-1 soil close to deficient limit < 10 mg kg-1 for

plant growth (Ahmed et al., 1994). Extractable Zn, Fe, Mn, Cu and B were

0.36, 3.53, 0.74, 0.19 and 0.33 mg kg-1, respectively. Zinc, Mn and B were

deficient, while Cu and Fe were adequate as reported by Zia et al. (2006).

4.2.2 Changes in Basic Soil Analyses during Incubation

Soil pH significantly decreased as recorded on 30, 60 and 90 days after

incubation (Table 12). No change was recorded with the isolates IW1 and IW14

and control. Maximum decrease in soil pH (net decrease of 0.78 points) was

recorded in case of inoculation IW16 with 50 mg kg-1 elemental S. Minimum

decrease in soil pH (net decrease of 0.03 points) was noted for the isolate SW2. It

was noted that Thiobacillus strains and S application alone could not reduce soil

pH remarkably but when they were used collectively a clear significant decrease

67

Table 11. Basic soil analyses before incubation

Soil property Unit Quantity

ECe dS m-1 1.02

pH - 7.90

O.M % 0.42

CaCO3 % 7.14

Total phosphorous mg kg-1 664.7

Available phosphorous mg kg-1 4.5

Total sulfur mg kg-1 214.5

Water soluble sulfur mg kg-1 9.6

Zinc mg kg-1 0.36

Iron mg kg-1 3.53

Manganese mg kg-1 0.74

Copper mg kg-1 0.19

Boron mg kg-1 0.33

Clay % 19.2

Sand % 71.4

Silt % 9.4

Textural class Sandy clay loam

68

Table 12. Changes in soil pH during incubation

Treatments

Soil pH Net Decrease in Soil pH

30-day 60-day 90-day 30-day 60-day 90-day

Control 7.90 a 7.90 a 7.90 a 0.00 0.00 0.00

IW16 7.86 ab 7.85 a-d 7.84 a-d 0.04 0.05 0.06

SW2 7.88 a 7.88 a-c 7.87 ab 0.02 0.02 0.03

IW1 7.90 a 7.90 ab 7.90 a 0.00 0.00 0.00

IW14 7.90 a 7.90 a 7.90 a 0.00 0.00 0.00

S1 7.88 a 7.87 a-d 7.85 a-c 0.02 0.03 0.05

S1 IW16 7.74 c-e 7.63 i 7.58 i 0.16 0.27 0.32

S1 SW2 7.80 a-c 7.70 gh 7.71fg 0.10 0.20 0.19

S1 IW1 7.83 a-c 7.78 ef 7.77 d-f 0.07 0.12 0.13

S1 IW14 7.85 ab 7.81 de 7.79 b-e 0.05 0.09 0.11

S2 7.86 ab 7.84 b-d 7.82 a-d 0.04 0.06 0.08

S2 IW16 7.66 e 7.51 j 7.49 j 0.24 0.39 0.41

S2 SW2 7.76 b-d 7.65 hi 7.60 hi 0.14 0.25 0.30

S2 IW1 7.80 a-c 7.74 fg 7.67 gh 0.10 0.16 0.23

S2 IW14 7.82 a-c 7.77 ef 7.74 e-g 0.08 0.13 0.16

S3 7.84 a-c 7.82 c-e 7.78 c-f 0.06 0.08 0.12

S3 IW16 7.52 e 7.35 k 7.12 l 0.38 0.55 0.78

S3 SW2 7.68 de 7.54 j 7.28 k 0.22 0.36 0.62

S3 IW1 7.74 c-e 7.67 hi 7.56 ij 0.16 0.23 0.34

S3 IW14 7.77 b-d 7.73 fg 7.67 gh 0.13 0.17 0.23

LSD 0.09 0.05 0.07

p ≥ F 0.05. Similar letter (s) values in a column are not statistically different: S1, 25 mg kg-1; S2, 37.5 mg kg-1; S3, 50 mg kg-1.

69

in pH was recorded. It was clear that S oxidation depended on both quantity of S

and efficient Thiobacillus strains. Combination of these two factors resulted in

maximum biological S oxidation which generated quantity of sulfuric acid and

reduce pH of the incubated soil. Stamford et al. (2003) and Stamford et al. (2007)

reported identical results and concluded that application of sulfur and

Acidithiobacilli in the soil significantly dropped soil pH as a consequent of H2SO4

production by biological S oxidation. Yang et al. (2010) also reported that S

application along with Thiobacillus spp decreased soil pH from 6.98 to 2.93 in 84

days due to biological S oxidation.

The highest increase in soil ECe (net increase of 1.35 dS m-1) was recorded

in treatment S3 IW16 (Thiobacillus strain IW16 plus 50 mg S kg-1) and the lowest

increase (net increase of 0.08 dS m-1) was found in treatment IW14 (Table 13).

The ECe of the incubated soil had a positive significant relationship with S

oxidation rate and huge negative significant association with soil pH. Treatments

wherein high sulfate contents were produced lowered the soil pH and ultimately

the concentration of plant available nutrients P, S and micronutrients increased

in the soil solution and hence the ECe of the incubated soil increased. These

findings were very much similar to the results reported by Garcia et al. (2007)

who concluded that biological S oxidation decreased soil pH and increased soil

EC during an incubation study of 100 days. Similar results were recorded by Oh et

al. (2010) during 84 days of incubation. They also reported a significant

relationship between the incubated soil EC and bacterial S oxidation rate.

70

Table 13. Soil electrical conductivity during the incubation

Treatments

Actual ECe Net increase in ECe


---------------------------------- dS m-1 ------------------------------------

Control 1.03 k 1.03 n 1.03 l 0.01 0.01 0.01

IW16 1.10 j 1.16 kl 1.18 jk 0.08 0.14 0.16

SW2 1.08 jk 1.14 klm 1.16 jk 0.06 0.12 0.14

IW1 1.07 jk 1.12 lm 1.13 k 0.05 0.10 0.11

IW14 1.06 jk 1.09 m 1.10 kl 0.04 0.07 0.08

S1 1.11 j 1.19 k 1.23 ij 0.09 0.17 0.21

S1 IW16 1.43 f 1.59 e 1.72 e 0.41 0.57 0.70

S1 SW2 1.32 g 1.46 g 1.61 f 0.30 0.44 0.59

S1 IW1 1.25 h 1.37 h 1.50 g 0.23 0.35 0.48

S1 IW14 1.19 i 1.28 ij 1.41 h 0.17 0.26 0.39

S2 1.17 i 1.24 j 1.28 i 0.15 0.22 0.26

S2 IW16 1.67 c 1.84 c 1.98 c 0.65 0.82 0.96

S2 SW2 1.59 d 1.68 d 1.81 d 0.57 0.66 0.79

S2 IW1 1.48 e 1.54 f 1.73 e 0.46 0.52 0.71

S2 IW14 1.36 g 1.45 g 1.61 f 0.34 0.43 0.59

S3 1.25 h 1.31 i 1.38 h 0.23 0.29 0.36

S3 IW16 1.91 a 2.18 a 2.37 a 0.89 1.16 1.35

S3 SW2 1.79 b 1.97 b 2.16 b 0.77 0.95 1.14

S3 IW1 1.65 c 1.80 c 1.94 c 0.63 0.78 0.92

S3 IW14 1.57 d 1.68 d 1.81 d 0.55 0.66 0.79

LSD 0.05 0.05 0.07

p ≥ F 0.05. Similar letter (s) values in a column are not statistically different. S1, 25 mg kg-1; S2, 37.5 mg kg-1; S3, 50 mg kg-1.

71

Table 14. Changes in soil CaCO3 contents during incubation

Treatments CaCO3 contents Net decrease in CaCO3


----------------------------------- % -------------------------------------------

Control 7.14 ab 7.14 ab 7.14 a 0.00 0.00 0.00

IW16 7.13 a-c 7.10 a-e 7.09 a-c 0.01 0.04 0.05

SW2 7.14 ab 7.12 a-d 7.11 ab 0.00 0.02 0.03

IW1 7.14 ab 7.13 a-c 7.13 a 0.00 0.01 0.01

IW14 7.14 a 7.14 a 7.14 a 0.00 0.00 0.00

S1 7.12 a-c 7.10 a-e 7.08 a-d 0.02 0.04 0.06

S1 IW16 7.06 a-e 7.03 d-g 7.01 e-g 0.08 0.11 0.13

S1 SW2 7.08 a-e 7.07 a-f 7.04 c-f 0.06 0.07 0.10

S1 IW1 7.10 a-d 7.08 a-f 7.06 b-e 0.04 0.06 0.08

S1 IW14 7.10 a-d 7.09 a-f 7.07 b-e 0.04 0.05 0.07

S2 7.10 a-d 7.09 a-f 7.07 b-e 0.04 0.05 0.07

S2 IW16 7.03 de 7.01 fg 6.98 gh 0.11 0.13 0.16

S2 SW2 7.05 b-e 7.04 d-g 7.02 e-g 0.09 0.10 0.12

S2 IW1 7.08 a-e 7.05 b-g 7.03 c-g 0.06 0.09 0.11

S2 IW14 7.08 a-e 7.07 a-f 7.05 c-e 0.06 0.07 0.09

S3 7.09 a-e 7.06 a-f 7.04 c-f 0.05 0.08 0.10

S3 IW16 7.00 e 6.97 g 6.93 h 0.14 0.17 0.21

S3 SW2 7.04 c-e 7.01 e-g 6.98 f-h 0.10 0.13 0.16

S3 IW1 7.06 a-e 7.04 c-g 7.01 e-g 0.08 0.10 0.13

S3 IW14 7.07 a-e 7.05 c-g 7.02 d-g 0.07 0.09 0.12

LSD 0.07 0.07 0.05


72

Maximum net decrease in CaCO3 contents was recorded as 0.21 % in

treatment where 50 mg S kg-1 soil was used along with Thiobacillus strain

IW16 (S3 IW16). Minimum decrease in CaCO3 contents was noted as 0.01 % in

treatment which contained Thiobacillus strain IW1 (Table 14). No decrease in

CaCO3 concentration was noted in control and in IW14. Amount of sulfuric acid

produced during biological S oxidation mechanism utilized in reaction with soil

calcite (CaCO3) and consequently gypsum was formed (Wandruszka, 2006).

Resultantly CaCO3 concentration decreased in the incubated soil during 0 to 30, 0

to 60 and 0 to 90 days of incubation period. However, the extent of decrease in

CaCO3 concentration depended on the nature of treatments. Results regarding

reduction in CaCO3 concentration in the incubated soil by S oxidation

corresponded with the results recorded by Garcia et al. (2007) during 100 days of

incubation study. They reported positive significant correlation between decrease

in soil pH and decrease in CaCO3 contents and recorded 10 to 20 % decrease in

CaCO3 contents in the soil with 1 to 2 points decrease in soil pH.

4.2.3 Soil Phosphorous Fractionation before Incubation

Data in Table 15 represent the quantities of different P fractions in soil

before incubation. Amongst the six P fractions determined in the fractionation

process, Ca2-P was the smallest fraction amounting 3.5 mg kg-1 (0.5 % of the total

soil P), while Ca10-P fraction was the biggest fraction with values of 307.3 mg kg-1

constituting 40.2 % of the total P and 65.0 % of the total inorganic P in the soil.

Total inorganic P (sum of all six fractions) was 472.6 mg kg-1 which constituted

73

Table 15. Soil phosphorous fractionation before incubation

Different Soil P fractions Quantity Percentage over

total soil P

(mg kg-1)

Ca2-P 3.5 0.5

Ca8-P 90.5 13.6

Al-P 28.5 4.3

Fe-P 24.3 3.7

O-P 18.5 2.8

Ca10-P 307.3 46.2

Total inorganic P (Sum of all six fractions)

472.6 71.1

Total calcium bounded P (sum of first, second and sixth fraction)

401.3 (84.9 % of the total

inorganic P) 60.4

Organic P (Total P minus total inorganic fractions)

192.3 28.9

Available P 4.5 0.7

Total P 664.7 -

74

71.0 % of the total P, out of which 401.3 mg kg-1 (84.9 % of the total inorganic P

and 60.4 % of the total soil P) was calcium bounded P. Total soil organic P was

calculated as 192.3 mg kg-1 (28.9 % of the total soil P). These results conform to

the findings by Memon et al. (2011).

Moreover, plant available P was determined separately and its

concentration in the soil was 4.5 mg kg-1 (0.7 % of the total soil P). Results

regarding P fractionation in calcareous soils reported by Mostashari et al. (2008)

were quite compatible with these results. They observed the amount of Ca2-P, Ca8-

P, Al-P, Fe-P, O-P and Ca10-P between 1.6 to 42.3 mg kg-1, 72 to 314 mg kg-1, 14.5

to 54.8 mg kg-1, 8.4 to 34.8 mg kg-1, 5.9 to 33.4 mg kg-1 and 262 to 697 mg kg-1 of

soil, respectively.

4.2.4 Changes in Soil Phosphorous Fractionation during Incubation

Data regarding different P fractions in soil after 30 days of incubation are

given in Table 16. Amongst the six P fractions in the incubated soil a considerable

increase in the amount of Ca2-P was recorded, while Ca8-P and Ca10-P fractions

decreased in all treatments except in control. However, no significant change was

noted in case of Al-P, Fe-P and O-P fractions. Maximum significant increase in

the quantity of Ca2-P (net increase of 9.1 mg kg-1) and maximum decrease in the

concentration of two P fractions Ca8-P and Ca10-P (net decrease of 6.0 and 1.9 mg

kg-1, respectively) was recorded in case of treatment S3 IW16 where maximum

dose of S 50 mg kg-1 soil was applied with strain IW16. Minimum increase in

75

Table 16. Soil phosphorous fractions after 30 days of incubation

Treatments Ca2-P Ca8-P Al-P Fe-P O-P Ca10-P

----------------------------------- mg kg-1-----------------------------------

Control 3.5 l 90.4 a 28.4 a 24.3 a 18.5 a 307.3 a

IW16 4.6 j 89.4 b 28.4 a 24.3 a 18.5 a 307.2 a

SW2 4.1 k 90.2 a 28.4 a 24.3 a 18.5 a 307.2 a

IW1 3.9 kl 90.3 a 28.5 a 24.3 a 18.5 a 307.3 a

IW14 3.8 kl 90.4 a 28.4 a 24.3 a 18.5 a 307.3 a

S1 3.9 kl 90.3 a 28.4 a 24.3 a 18.5 a 307.3 a

S1 IW16 7.3 e 87.6 ef 28.2 a 24.2 a 18.6 a 306.8 ab

S1 SW2 6.1 gh 88.5 cd 28.3 a 24.2 a 18.5 a 307.0 ab

S1 IW1 5.3 i 88.9 bc 28.3 a 24.3 a 18.3 a 307.3 a

S1 IW14 5.3 i 89.1 bc 28.4 a 24.3 a 18.5 a 307.3 a

S2 5.2 i 89.1 bc 28.5 a 24.2 a 18.5 a 307.3 a

S2 IW16 9.6 c 86.7 g 28.1 a 24.1 a 18.1 a 305.8 c

S2 SW2 7.8 d 87.2 fg 28.1 a 24.1 a 18.5 a 306.8 ab

S2 IW1 6.8 f 87.4 e-g 28.3 a 24.3 a 18.5 a 307.2 a

S2 IW14 5.9 h 88.5 cd 28.5 a 24.2 a 18.5 a 307.2 a

S3 6.2 g 88.1 de 28.3 a 24.2 a 18.3 a 307.1 a

S3 IW16 12.6 a 84.5 i 28.1 a 23.9 a 17.9 a 305.4 c

S3 SW2 10.2 b 85.7 h 28.2 a 24.0 a 18.1 a 306.1 bc

S3 IW1 7.7 d 87.2 fg 28.2 a 24.10 a 18.2 a 306.9 ab

S3 IW14 6.7 f 87.8 d-f 28.4 a 24.16 a 18.3 a 307.1 ab

LSD 0.3 0.7 1.2 1.1 0.8 0.8


76



----------------------------------- mg kg-1----------------------------------

Control 3.5 o 90.4 a 28.4 a 24.3 a 18.5 a 307.4 a

IW16 7.1 i 87.7 c 28.3 a 24.3 a 18.5 a 306.7 a-e

SW2 5.9 k 88.7 b 28.3 a 24.3 a 18.5 a 307.0 a-d

IW1 5.2 m 89.1 b 28.5 a 24.3 a 18.5 a 307.1 a-c

IW14 4.7 n 89.5 b 28.5 a 24.3 a 18.5 a 307.2 ab

S1 4.8 n 89.3 b 28.5 a 24.3 a 18.6 a 307.2 ab

S1 IW16 9.8 d 86.5 de 28.1 a 24.1 a 18.5 a 305.7 f-h

S1 SW2 7.0 i 87.6 c 28.2 a 24.1 a 18.5 a 306.2 c-g

S1 IW1 6.1 j 88.7 b 28.3 a 24.2 a 18.5 a 306.9 a-e

S1 IW14 5.8 k 88.7 b 28.4 a 24.3 a 18.5 a 307.0 a-d

S2 5.7 l 88.9 b 28.4 a 24.2 a 18.5 a 307.2 a-c

S2 IW16 11.9 c 85.2 fg 27.9 a 24.0 a 18.4 a 305.4 gh

S2 SW2 9.8 d 86.4 de 27.9 a 24.1 a 18.5 a 305.9 e-h

S2 IW1 8.3 f 86.9 cd 28.1 a 24.2 a 18.5 a 306.6 a-f

S2 IW14 8.1 g 87.0 cd 28.2 a 24.2 a 18.5 a 306.7 a-e

S3 7.8 h 87.7 c 28.2 a 24.1 a 18.4 a 306.4 b-f

S3 IW16 16.6 a 82.3 h 26.7 a 23.7 a 18.2 a 304.1 i

S3 SW2 12.8 b 84.7 g 27.9 a 23.9 a 18.2 a 305.1 h

S3 IW1 9.8 d 85.9 ef 28.0 a 24.0 a 18.4 a 306.1 e-g

S3 IW14 8.7 e 86.9 cd 28.1 a 24.1 a 18.4 a 306.1 d-g

LSD 0.1 0.8 1.6 1.1 0.7 0.8


77



------------------------------------- mg kg-1------------------------------------

Control 3.5 n 90.5 a 28.5 a 24.3 a 18.6 a 307.4 a

IW16 7.8 i 87.2 de 28.1 ab 24.1 a-c 18. ab 306.7 a-c

SW2 6.2 k 88.4 c 28.3 ab 24.3 ab 18.2 ab 307.0 ab

IW1 5.3 l 89.0 bc 28.4 ab 24.1 a-c 18.5 ab 307.0 ab

IW14 4.9 m 89.1 bc 28.2 ab 24.2 a-c 18.2 ab 307.2 ab

S1 5.3 l 89.2 b 28.5 ab 24.3 ab 18.5 ab 307.1 ab

S1 IW16 12.6 d 86.0 fg 27.6 a-c 23.2 b-e 18.2 ab 304.8 fg

S1 SW2 8.6 g 87.4 d 28.1 ab 23.7 a-e 18.4 ab 306.1 c-e

S1 IW1 6.8 j 88.4 bc 28.1 ab 23.8 a-d 18.5 ab 306.7 a-c

S1 IW14 6.2 k 88.5 bc 28.2 ab 23.9 a-d 18.5 ab 306.9 a-c

S2 6.2 k 88.6 bc 28.3 ab 24.1 a-c 18.5 ab 307.0 ab

S2 IW16 15.8c 84.8 h 26.8 bc 22.6 ef 17.7 bc 304.4 g

S2 SW2 12.5 d 85.6 gh 27.5 a-c 23.1 a-c 18.2 ab 304.8 fg

S2 IW1 10.8 e 86.5 ef 27.9 ab 23.4 a-e 18.5 ab 305.4 ef

S2 IW14 8.5 g 86.7 d-f 28.1 ab 23.6 a-e 18.5 ab 306.6 a-c

S3 8.3 h 87.2 de 28.1 ab 23.6 a-e 18.3 ab 306.3 b-d

S3 IW16 23.9 a 80.3 j 26.1 c 22.1 f 17.1 c 302.6 h

S3 SW2 17.0 b 82.5 i 27.2 a-c 22.7 ef 17.8 a-c 304.4 g

S3 IW1 12.6 d 85.5 gh 27.7 ab 22.9 d-f 18.1 ab 305.1 fg

S3 IW14 10.2 f 86.3 fg 27.9 ab 23.1 c-e 18.2 ab 305.5 d-f

LSD 0.1 0.7 1.4 0.9 0.7 0.8


78

Ca2-P fraction (net increase of 0.3 mg kg-1) was recorded in treatment where

Thiobacilli strain IW14 was inoculated without S. No change in any P fraction

was observed in control.

Dissolution trend of insoluble calcium bounded P compounds continued

upto 60 days of incubation and the data are presented in Table 17 which predicted

that S oxidation process was going on. Significant increase in Ca2-P concentration

(net increase of 13.1 mg kg-1) and decrease (dissolution) in the amount of

insoluble P fractions Ca8-P and Ca10-P (net decrease of 8.2 and 3.2 mg kg-1,

respectively) was noted with S3 IW16 (50 mg S kg-1 soil with strain IW16). No

significant change was found in the quantity of Al-P, Fe-P and O-P fractions in all

the treatments. Similarly, no change was noted in the concentration of all the six

soil P fractions in control treatment. The treatment having strain IW14 alone

predicted the lowest increase in Ca2-P quantity (net increase of 1.2 mg kg-1) and

the lowest decrease in Ca8-P and Ca10-P contents (net decrease of 1.0 and 0.1 mg

kg-1, respectively). The highly insoluble P compounds in soil (Ca8-P and Ca10-P)

were effectively solubilizing into soluble and plant available P compound (Ca2-P)

under the effect of Thiobacilli spp. (IW16 and SW2) in the presence of maximum

dose of 50 mg S kg-1 soil.

Increased concentration of bacterially produced sulfuric acid in the

incubated soil carried on the mechanism of P dissolution upto the 90th day of

incubation (Table 18). Concentration of three P fractions Fe-P, Al-P and O-P

79

showed non significant change in all the treatments except with S3 IW16 where

small decrease in these fractions was observed. Treatment S3 IW16 (50 mg S kg-1

soil with IW16 strain) contained the highest net increased amount of Ca2-P (20.4

mg kg-1) and the highest net reduced values of Ca8-P and Ca10-P fractions (10.2

and 4.7 mg kg-1, respectively). The smallest net increase in Ca2-P and smallest net

decrease in Ca8-P and Ca10-P was observed in IW14. No significant change in

different soil P fractions was noted in control.

The efficient sulfuric acid producers (IW16 and SW2) in combination with

50 mg S kg-1 dissolved insoluble calcium bounded P fractions Ca8-P and Ca10-P

into sparingly soluble plant available Ca2-P fraction. Phosphorous solubilization

and enhancement in soil was highly linked with the combination of both

Thiobacillus strains and S application as reported by Bhatti and Yawar (2010).

Results described by Lee et al. (2004) were similar with these results who

concluded that microbial activities in the soil converted insoluble P fractions into

bio-available P fractions in soil. Stamford et al. (2003) and Stamford et al. (2007)

reported that Acidithiobacillus species produced sulfuric acid by S application in

soil, which enhanced bioavailability of P in soil.

80

y = -2.6241x + 21.821

R2 = 0.89

1.0

1.3

1.6

1.9

2.2

2.5

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

EC

e (d

S m

-1) y = 0.3886x + 4.0564

R2 = 0.89

6.9

7.0

7.1

7.2

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Soi

l CaC

O3

cont

ents

(%

)

y = -2.0879x + 17.621

R2 = 0.92

1.0

1.3

1.6

1.9

2.2

2.5

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

EC

e (d

S m

-1) y = 0.2884x + 4.838

R2 = 0.90

6.9

7.0

7.1

7.2

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Soi

l CaC

O3

cont

ents

(%

)

y = -1.7387x + 14.931

R2 = 0.91

1.0

1.3

1.6

1.9

2.2

2.5

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

EC

e (d

S m

-1) y = 0.2452x + 5.1642

R2 = 0.85

6.9

7.0

7.1

7.2

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Soi

l CaC

O3

cont

ents

(%

)

Figure 5. Soil pH relation with (a) ECe and (b) CaCO3 contents changes during

incubation: a1 to a3 and b1 to b3 represent data from day 30, 60 and 90,

respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

81

y = -24.185x + 194.94

R2 = 0.97

0

6

12

18

24

30

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Ca2

-P (

mg

kg-1

) y = -57.325x + 412.59

R2 = 0.92

0

6

12

18

24

30

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Ca2

-P (

mg

kg-1

)

y = -20.543x + 167.01

R2 = 0.93

0

6

12

18

24

30

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Ca2

-P (

mg

kg-1

)

y = -66.4x + 477.45

R2 = 0.90

0

6

12

18

24

30

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Ca2

-P (

mg

kg-1

)

y = -23.364x + 189.37

R2 = 0.96

0

6

12

18

24

30

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Ca2

-P (

mg

kg-1

) y = -82.67x + 592.51

R2 = 0.85

0

6

12

18

24

30

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Ca2

-P (

mg

kg-1

)

Figure 6. Relation of Ca2-P with (a) soil pH and (b) CaCO3 changes during

incubation: a1 to a3 and b1 to b3 represent data from day 30, 60 and 90, respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

82

y = -23.944x + 194.22

R2 = 0.92

0

6

12

18

24

30

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Bio

avai

labl

e P

(m

g kg-1

)y = -56.31x + 406.54

R2 = 0.86

0

6

12

18

24

30

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Bio

avai

labl

e P

(m

g kg-1

)

y = -23.348x + 189.82

R2 = 0.94

0

6

12

18

24

30

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Bio

avai

labl

e P

(m

g kg-1

)

y = -75.275x + 541.29

R2 = 0.91

0

6

12

18

24

30

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Bio

avai

labl

e P

(m

g kg-1

)

y = -27.045x + 218.82

R2 = 0.98

0

6

12

18

24

30

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Bio

avai

labl

e P

(m

g kg-1

)

y = -93.559x + 670.42

R2 = 0.83

0

6

12

18

24

30

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Bio

avai

labl

e P

(m

g kg-1

)

Figure 7. Bio-available P relation with (a) soil pH and (b) CaCO3 contents changes

during incubation: a1 to a3 and b1 to b3 represent data from day 30, 60 and 90,

respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

83

y = 16.085x - 37.1

R2 = 0.91

75

79

83

87

91

95

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Ca8

-P (

mg

kg-1

)

y = 39.523x - 191.75

R2 = 0.93

7579

8387

9195

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Ca8

-P (

mg

kg-1

)

y = 12.331x - 8.0669

R2 = 0.91

75

79

83

87

91

95

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Ca8

-P (

mg

kg-1

)

y = 40.187x - 196.74

R2 = 0.90

757983879195

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Ca8

-P (

mg

kg-1

)

y = 11.192x + 0.7887

R2 = 0.94

75

79

83

87

91

95

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Ca8

-P (

mg

kg-1

)

y = 39.474x - 191.43

R2 = 0.83

757983879195

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Ca8

-P (

mg

kg-1

)

Figure 8. Relation of Ca8-P with (a) soil pH and (b) CaCO3 contents changes


respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

84

y = 5.2867x + 265.71

R2 = 0.88

300

302

304

306

308

310

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Ca1

0 -P

(mg

kg-1

)

y = 11.368x + 226.38

R2 = 0.69

300302304306308310

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Ca1

0 -P

(m

g kg

-1)

y = 5.518x + 263.69

R2 = 0.95

300

302

304

306

308

310

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Ca1

0 -P

(mg

kg-1

)

y = 17.368x + 183.62

R2 = 0.87

300302304306308310

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Ca1

0 -P

(m

g kg

-1)

y = 5.8288x + 261.11

R2 = 0.92

300

302

304

306

308

310

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Ca1

0 -P

(mg

kg-1

)

y = 21.099x + 157.19

R2 = 0.86

300302304306308310

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Ca1

0 -P

(m

g kg

-1)

Figure 9. Ca10-P relation with (a) soil pH and (b) CaCO3 contents changes during


respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

85

Table 19. Enhancement in soil phosphorous bioavailability during incubation

Treatments Bio-available P

Net increase in bio-available P

30-day 60-day 90-day 30- day

60- day

90-day

----------------------------------mg kg-1-------------------------------

Control 4.5 j 4.5 m 4.5 p 0.0 0.0 0.0

IW16 6.6 g 7.9 h 8.3 j 2.1 3.4 3.8

SW2 5.7 hi 6.5 ij 6.9 m 1.2 2.0 2.4

IW1 5.1 ij 5.8 k 5.9 n 0.6 1.3 1.4

IW14 4.9 j 5.1 l 5.3 o 0.4 0.6 0.8

S1 5.2 ij 5.6 k 6.0 n 0.7 1.1 1.5

S1 IW16 9.5 c 11.2 e 13.8 e 5.0 6.7 9.3

S1 SW2 7.2 f 8.4 g 9.3 h 2.7 3.9 4.8

S1 IW1 6.1 gh 6.8 i 7.7 k 1.6 2.3 3.2

S1 IW14 5.7 hi 6.3 j 6.9 lm 1.2 1.8 2.4

S2 6.1 gh 6.7 i 7.l 1.6 2.2 2.5

S2 IW16 11.0 b 13.9 c 16.7 c 6.5 9.4 12.2

S2 SW2 9.5 c 11.1 e 13.8 e 5.0 6.6 9.3

S2 IW1 7.3 f 9.4 f 11.1 g 2.8 4.9 6.6

S2 IW14 6.3 gh 8.8 g 9.1 i 1.8 4.3 4.6

S3 8.1 e 8.8 g 9.1 i 3.6 4.3 4.6

S3 IW16 14.3 a 21.4a 26.8 a 9.8 16.9 22.3

S3 SW2 11.2 b 14.6 b 21.3 b 6.7 10.1 16.8

S3 IW1 8.8 d 11.7 d 14.0 d 4.3 7.2 9.5

S3 IW14 7.2 f 9.7 f 12.1 f 2.7 5.2 7.6

LSD 0.6 0.4 0.2


86

4.2.5 Phosphorous Bioavailability Enhancement during Incubation

The amount of bio-available P in soil increased in different treatments

during incubation period but the highest and the most significant increase was

recorded in treatment S3 IW16 with net increased value of 22.3 mg kg-1 (Table 19).

The lowest net increase in the quantity of soil available P was recorded as 0.8 mg

kg-1 with IW14 strain without S application. No significant change in the amount

of soil available P was noted in control during the whole period of incubation.

Enhancement of soil bio-available P contents was associated with the

quantity of S and the efficiency of Thiobacillus strains inoculated. Best

combination of these two factors helped in generating sulfuric acid as a result of

bacterial S oxidation which solubilized complex insoluble calcium bounded P

compounds into simple soluble plant available P forms. Similarly, Besharati et al.

(2007) also reported a significant increase in plant available P in soil through

combined application of S and Thiobacillus. Stamford et al. (2003) reported that

the application of elemental S with Thiobacillus was highly significant in P

solubilization from apatite. Kumar and Nagendran (2008) also reported P

solubilization by the combined effect of S and Thiobacillus strain

Acidithiobacillus Thiooxidans.

87

Table 20. Soil sulfur fractionation before incubation

Different Soil S fractions Quantity Percentage

over total S mg kg-1

S-H2O 9.6 4.5

S-NaH2PO4 4.4 2.0

S-HCL 7.6 3.5

Mineral S (sum of first three fractions)

21.6 10.1

Organic S (Total S minus Total inorganic S)

192.9 90.0

Total S 214.5

88

4.2.6 Soil Sulfur Fractionation before Incubation

The concentration of water soluble S, sorbed S, occluded S, mineral S,

organic S and total S in the soil was 9.6 (4.5 % of the total S), 4.4 (2.1 % of the

total S), 7.6 (3.5 % of the total S), 21.6 (10.1 % of the total S), 192.9 (90.0 % of

the total S) and 214.5 mg kg-1, respectively (Table 20). Balik et al. (2007) and

Morche (2008) determined total soil S between 0.01 to 0.1 % and the

concentration of total S (0.02 %) determined in the present study was recorded in

the similar range. The results regarding organic S were confirmed by Hu et al.

(2005) who estimated organic S as a dominant and major fraction in calcareous

soils. These results were further verified by various scientists (Chapmann, 2001;

Solomon et al., 2001; Yang et al., 2007b) who reported soil organic S upto 95 %

of the total S in soil. Balik et al. (2009) studied quantities of different S fractions

in soil and their results regarding water soluble, sorbed and occluded S were

agreed with these results.

4.2.7 Changes in Soil Sulfur Fractionation during Incubation

Treatment S3 IW16 enclosed the highest significant net increase in the

quantities of water soluble S (S-H2O) and sorbed S (S-NaH2PO4) as 16.6 and 0.8

mg kg-1, respectively and the highest net decreased value of occluded S (S-HCl) as

0.8 mg kg-1 after 30 days of incubation (Table 21). The lowest change in the

quantities of S fractions was noted with IW14 where net increase in the quantity of

water soluble S was recorded as 0.1 mg kg-1 soil. The incubated soil in control

treatment predicted no significant change in all fractions of S in the soil.

89

Table 21. Changes in sulfur fractions 30 days after incubation

Treatments S-H2O S-NaH2PO4 S-HCl

--------------------------- mg kg-1-------------------------

Control 9.6 m 4.4 ij 7.6 a

IW16 11.3 j 4.4 ij 7.6 a

SW2 10.1 l 4.4 ij 7.6 a

IW1 9.8 lm 4.4 ij 7.6 a

IW14 9.7 lm 4.4 ij 7.6 a

S1 10.0 lm 4.4 ij 7.6 a

S1 IW16 15.6 fg 4.8 c-e 7.5 a

S1 SW2 14.3 h 4.6 fg 7.5 a

S1 IW1 12.7 i 4.5 h-j 7.5 a

S1 IW14 10.8 k 4.4 ij 7.6 a

S2 11.5 j 4.5 h-j 7.6 a

S2 IW16 20.9 c 4.9 b 7.4 ab

S2 SW2 17.4 e 4.8 b-d 7.5 a

S2 IW1 15.3 g 4.7 d-f 7.5 a

S2 IW14 12.5 i 4.6 fg 7.5 a

S3 12.6 i 4.6 fg 7.6 a

S3 IW16 26.2 a 5.2 a 6.8 c

S3 SW2 22.7 b 4.9 b 7.1 b

S3 IW1 18.7 d 4.8 c-e 7.4 ab

S3 IW14 15.8 f 4.7 d-f 7.4 a

LSD 0.4 0.1 0.2


90



---------------------------- mg kg-1-------------------------

Control 9.6 s 4.4 ij 7.6 a

IW16 14.3 k 4.5 hi 7.4 a-d

SW2 12.2 o 4.4 ij 7.6 a

IW1 10.5 q 4.4 ij 7.6 a

IW14 9.9 r 4.4 ij 7.6 a

S1 11.2 p 4.4 ij 7.6 a

S1 IW16 20.5 f 4.9 d 7.3 a-d

S1 SW2 17.5 i 4.8 e 7.3 a-d

S1 IW1 14.2 l 4.7 fg 7.4 a-d

S1 IW14 12.5 n 4.5 h 7.5 ab

S2 13.4 m 4.5 h 7.6 a

S2 IW16 26.6 b 5.2 c 7.1 c-e

S2 SW2 22.5 d 4.9 d 7.3 a-d

S2 IW1 18.5 g 4.8 e 7.5 a-c

S2 IW14 15.8 j 4.7 ef 7.5 ab

S3 13.4 m 4.6 gh 7.5 ab

S3 IW16 33.6 a 5.7 a 6.5 f

S3 SW2 25.2 c 5.4 b 6.9 e

S3 IW1 21.4 e 5.2 c 7.1 de

S3 IW14 17.7 h 5.2 c 7.2 b-e

LSD 0.1 0.1 0.3

p = 0.05. Similar letter (s) values in a column are not statistically different. S1, 25 mg kg-1; S2, 37.5 mg kg-1; S3, 50 mg kg-1.

91



----------------------------- mg kg-1---------------------------

Control 9.6 s 4.4 k 7.6 a

IW16 15.3 l 4.6 gh 7.3 b-d

SW2 13.5 o 4.5 hi 7.5 ab

IW1 11.3 q 4.5 hi 7.5 a

IW14 10.2 r 4.4 jk 7.6 a

S1 12.4 p 4.5 hi 7.6 a

S1 IW16 24.5 f 5.3 d 7.1 d-f

S1 SW2 20.6 h 5.1 e 7.1 d-f

S1 IW1 16.7 j 4.8 f 7.2 cd

S1 IW14 14.8 m 4.7 g 7.3 b-d

S2 14.4 n 4.6 g 7.5 a

S2 IW16 31.5 c 5.7 b 6.8 fg

S2 SW2 25.9 d 5.5 c 7.1 d-f

S2 IW1 22.5 g 5.1 e 7.3 b-d

S2 IW14 17.5 i 4.9 f 7.4 a-c

S3 15.6 k 4.7 g 7.4 a-c

S3 IW16 40.2 a 6.1 a 6.3 h

S3 SW2 32.7 b 5.7 b 6.6 g

S3 IW1 24.7 e 5.4 c 6.9 e-g

S3 IW14 20.6 h 5.3 d 7.1 d-f

LSD 0.1 0.1 0.2


92

After 60 days of incubation net increased quantities of water soluble S and

exchangeable S were 24.0 and 1 .3 mg kg-1, respectively (Table 22). However, the

quantity of occluded S decreased (net decrease 1.1 mg kg-1) due to its dissolution

and conversion to water soluble and exchangeable S forms. Minimum increase

(0.3 mg kg-1) was recorded in the quantity of water soluble S with IW14.

Concentration of all S fractions in control changed non significantly.

Mechanism of sulfate production by interactive effect of different doses of

S and Thiobacillus spp. continued upto the 90th day of incubation (Table 23).

Maximum net increase in the concentration of water soluble S and exchangeable S

was recorded as 30.6 and 1.7 mg kg-1, respectively and maximum net decrease in

the amount of occluded S was determined as 1.3 mg kg-1 with S3 IW16. Decrease

in occluded S was due to its solubilization by high quantity of sulfuric acid

produced as a result of S oxidation. The treatment IW14 indicated minimum net

increase in the quantity of water soluble S as 0.6 mg kg-1 No significant change

was observed in the amount of all S fractions in case of control.

Sulfur or Thiobacillus spp. alone could not produce significant amount of

sulfates, whereas S along with Thiobacillus strains significantly increased the

concentration of water soluble S and exchangeable S in the incubated soil because

both are essential for bacterial S oxidation phenomenon (Jiang et al., 2009; Hassan

et al., 2010). The results were consistent with the results reported by Yang et al.

(2010) regarding elemental S oxidation by Thiobacillus spp. wherein they

93

recorded an increase in the concentration of both water soluble S and

exchangeable S upto 84th day of incubation. Maochun et al. (2002) and Kumar and

Nagendran (2008) both recorded sulfate production during S oxidation under the

combined effect of S and Acidithiobacillus thiooxidans.

4.2.8 Micronutrients Bioavailability Enhancement during Incubation

After 60 days of incubation maximum quantities of Zn (1.43 mg kg-1), Fe

(7.15 mg kg-1), Mn (2.88 mg kg-1), Cu (0.48 mg kg-1) and B (1.00 mg kg-1) with S3

IW16 in which the soil was treated with maximum amount of 100 mg S kg-1 in

combination with Thiobacillus strain IW16 (Table 24). Minimum concentration of

Zn, Fe, Mn, Cu and B (0.37, 3.55, 0.73, 0.20 and 0.36 mg kg-1, respectively) was

noted in treatment where only strain IW14 was inoculated. No significant change

was found in control.

Table 25 shows the data regarding the concentration of soil micronutrients

after 60 days of incubation. An increase in Zn, Fe, Mn, Cu and B quantities was

observed in all treatments except in control where the soil was incubated without S

and Thiobacilli. The highest concentration of Zn, Fe, Mn, Cu and B in the

incubated soil was observed as 1.65, 8.94, 3.73, 0.64 and 1.26 mg kg-1,

respectively in treatment S3 IW16. The lowest quantities of Zn, Fe, Mn, Cu and B

were recorded in treatment IW14 with values of 0.38, 3.69, 0.78, 0.21 and 0.37 mg

kg-1, respectively.

94

Table 24. Soil micronutrients contents after 30 days of incubation

Treatments Zn Fe Mn Cu B

---------------------------------- mg kg-1-------------------------------

Control 0.37 i 3.54 h 0.75 i 0.20 g 0.34 h

IW16 0.39 i 3.67 f-h 0.88 e-h 0.22 fg 0.44 fg

SW2 0.38 i 3.58 h 0.80 g-i 0.21 g 0.38 h

IW1 0.38 i 3.57 h 0.78 hi 0.20 g 0.37 h

IW14 0.37 i 3.55 h 0.73 i 0.20 g 0.36 h

S1 0.38 i 3.58 h 0.77 hi 0.20 g 0.36 h

S1 IW16 0.68 e 3.95 e 1.15 d 0.25 e-g 0.55 e

S1 SW2 0.51 fg 3.79 e-g 0.96 e 0.23 fg 0.47 f

S1 IW1 0.48 gh 3.68 f-h 0.82 f-i 0.21 fg 0.39 gh

S1 IW14 0.43 hi 3.62 gh 0.78 hi 0.21 g 0.38 h

S2 0.44 hi 3.67 f-h 0.80 g-i 0.21 fg 0.38 h

S2 IW16 0.93 c 5.54 b 1.53 b 0.37 b 0.80 b

S2 SW2 0.78 d 4.15 d 1.10 d 0.32 bc 0.67 c

S2 IW1 0.67 e 3.94 e 0.92 ef 0.27 c-f 0.58 de

S2 IW14 0.59 f 3.83 ef 0.84 f-i 0.26 d-g 0.49 f

S3 0.53 fg 3.80 e-g 0.89 e-g 0.25 e-g 0.44 fg

S3 IW16 1.43 a 7.15 a 2.88 a 0.48 a 1.00 a

S3 SW2 1.15 b 5.52 b 1.57 b 0.36 b 0.80 b

S3 IW1 0.89 c 4.59 c 1.37 c 0.31 b-d 0.62 cd

S3 IW14 0.78 d 4.14 d 1.16 d 0.29 c-e 0.60 de

LSD 0.07 0.17 0.09 0.05 0.05


95



--------------------------------- mg kg-1----------------------------------

Control 0.37 i 3.54 m 0.76 l 0.20 k 0.33 m

IW16 0.43 hi 4.14 i 0.97 i 0.26 g-k 0.52 ij

SW2 0.40 i 3.87 jk 0.87 i-l 0.23 h-k 0.42 k-m

IW1 0.39 i 3.76 kl 0.83 j-l 0.22 h-k 0.38 lm

IW14 0.38 i 3.69 l 0.78 kl 0.21 jk 0.37 lm

S1 0.42 hi 3.73 l 0.79 kl 0.21 i-k 0.39 lm

S1 IW16 0.82 e 4.76 gh 1.36 g 0.33 e-g 0.69 ef

S1 SW2 0.69 f 4.11 i 1.16 h 0.29 f-i 0.60 gh

S1 IW1 0.61 g 3.98 j 0.94 ij 0.26 g-k 0.47 jk

S1 IW14 0.57 g 3.89 jk 0.87 i-l 0.25 g-k 0.45 j-l

S2 0.48 h 4.17 i 0.88 i-k 0.24 h-k 0.40 k-m

S2 IW16 1.29 c 6.53 c 2.87 b 0.48 bc 0.79 cd

S2 SW2 0.92 d 5.16 f 1.94 d 0.40 de 0.79 cd

S2 IW1 0.82 e 4.89 g 1.69 e 0.33 e-g 0.64 fg

S2 IW14 0.71 f 4.64 h 1.49 f 0.30 f-h 0.59 g-i

S3 0.58 g 4.80 g 0.95 i 0.28 g-j 0.53 h-j

S3 IW16 1.65 a 8.94 a 3.73 a 0.64 a 1.26 a

S3 SW2 1.53 b 7.15 b 2.44 c 0.51 b 0.92 b

S3 IW1 1.26 c 6.16 d 1.98 d 0.42 cd 0.86 bc

S3 IW14 0.97 d 5.35 e 1.89 d 0.37 d-f 0.75 de

LSD 0.07 0.13 0.10 0.07 0.07


96



---------------------------------- mg kg-1-------------------------------

Control 0.37 p 3.54 n 0.75 l 0.20 k 0.34 n

IW16 0.46 n 4.58 i 1.12 i 0.27 i-k 0.56 jk

SW2 0.41 op 4.31 j 0.94 j 0.24 jk 0.46 lm

IW1 0.39 p 4.06 kl 0.89 jk 0.22 k 0.39 mn

IW14 0.38 p 3.88 m 0.80 kl 0.21 k 0.38 mn

S1 0.46 no 3.92 lm 0.84 j-l 0.22 k 0.39 mn

S1 IW16 1.25 h 5.72 f 1.65 g 0.37 f-h 0.77 fg

S1 SW2 0.95 j 4.98 h 1.43 h 0.34 g-i 0.68 hi

S1 IW1 0.79 k 4.54 i 1.19 i 0.32 h-j 0.61 ij

S1 IW14 0.69 l 4.13 k 1.10 i 0.31 h-j 0.49 kl

S2 0.53 m 4.30 j 0.95 j 0.28 i-k 0.48 kl

S2 IW16 1.76 c 8.54 b 3.66 b 0.68 b 1.13 b

S2 SW2 1.60 e 6.91 d 2.83 d 0.51 cd 0.99 c

S2 IW1 1.37 g 6.11 e 2.16 e 0.49 cd 0.85 ef

S2 IW14 1.15 i 5.22 g 1.93 f 0.44 d-f 0.74 gh

S3 0.68 l 4.92 h 1.15 i 0.41 e-g 0.62 ij

S3 IW16 2.15 a 10.13 a 4.53 a 0.79 a 1.63 a

S3 SW2 1.82 b 8.24 c 3.16 c 0.68 b 1.12 b

S3 IW1 1.70 d 6.82 d 2.91 d 0.55 c 0.92 cd

S3 IW14 1.54 f 6.15 e 2.18 e 0.48 c-e 0.87 de

LSD 0.05 0.15 0.12 0.07 0.07


97

Increasing trend of micronutrients concentration in the incubated soil

continued up to the 90th day of incubation (Table 25). After 90 days of incubation

the highest values of Zn (2.15 mg kg-1), Fe (10.13 mg kg-1), Mn (4.53 mg kg-1), Cu

(0.79 mg kg-1) and B (1.63 mg kg-1) in the incubated soil were recorded with S3

IW16 (Table 26). While the minimum quantities of soil micronutrients Zn (0.38

mg kg-1), Fe (3.88 mg kg-1), Mn (0.80 mg kg-1), Cu (0.21 mg kg-1) and B (0.38 mg

kg-1) were determined with IW14. Amongst the treatments control showed no

significant difference in the quantities of soil micronutrients.

During 90 days of incubation period bacterial S oxidation continuously

produced sulfuric acid which persistently solubilized and released soil

micronutrients (Zn, Fe, Mn, Cu and B) into available forms as was reported by

Jaggi et al. (2005). Similar results were recorded by Zhou et al. (2002) during

their study on S oxidation by Thiobacillus. They reported that biological S

oxidation was a sulfuric acid generating process which not only decreased soil pH

but also increased the availability of soil micronutrients. Yang et al. (2010) also

reported that the availability of soil micronutrients increased during the

mechanism of biological S oxidation by Thiobacillus spp during 84 days of

incubation study.

4.2.9 Interrelationship among Various Soil Variables

Degree of linear relationship among various pairs of variables during

incubation study (after 30, 60 and 90 days) was determined through simple linear

98

Table 27. Correlation among various soil variables

Correlation parameters pH ECe CaCO3

After 30 days

EC -0.94**

CaCO3 0.95** -0.96**

Ca2-P -0.98** 0.96** -0.96**

Ca8-P 0.95** -0.97** 0.96**

Ca10-P 0.94** -0.70** 0.68**

BAP -0.97** 0.92** -0.93**

Zn -0.97** 0.97** -0.92**

Fe -0.95** 0.86** -0.83**

Mn -0.94** 0.83** -0.81**

Cu -0.96** 0.94** -0.90**

B -0.97** 0.96** -0.92**

After 60 days

EC -0.96**

CaCO3 0.95** -0.96**

Ca2-P -0.96** 0.95** -0.95**

Ca8-P 0.95** -0.96** 0.95**

Ca10-P 0.97** -0.90** 0.91**

BAP -0.97** 0.96** -0.95**

Continued

99

Table Page 2

Correlation parameters pH ECe CaCO3

Zn -0.95** 0.95** 0.98**

Fe -0.93** 0.94** -0.90**

Mn -0.95** 0.94** -0.89**

Cu -0.96** 0.97** -0.93**

B -0.95** 0.97** -0.93**

After 90 days

EC -0.95**

CaCO3 0.92** -0.96**

Ca2-P -0.98** 0.94** -0.92**

Ca8-P 0.97** -0.93** 0.91**

Ca10-P 0.96** -0.92** 0.91**

BAP -0.99** 0.94** -0.91**

Zn -0.92** 0.98** -0.92**

Fe -0.96** 0.95** -0.91**

Mn -0.94** 0.94** -0.89**

Cu -0.94** 0.96** -0.93**

B -0.96** 0.96** -0.94**

* p ≥ F 0.05 ** p ≥ F 0.01 BAP: Bio-available phosphorous

100

Table 28. Linear regression analyses among various soil variables

Variables Linear regression equation Coefficient of determination

(R2)EC vs. pH after 30 days y = -2.6241 pH + 21.821 0.89

EC vs. pH after 60 days y = -2.0879 pH + 17.621 0.92

EC vs. pH after 90 days y = -1.7387 pH + 14.931 0.91

CaCO3 vs. pH after 30 days y = 0.3886 pH + 4.0564 0.89



Ca2-P vs. pH after 30 days y = -24.185 pH + 194.94 0.97



Ca2-P vs. CaCO3 after 30 days y = -57.325 CaCO3 + 412.59 0.92



Ca8-P vs. pH after 30 days y = 16.085 pH - 37.1 0.91

Ca8-P vs. pH after 60 days y = 12.331 pH - 8.0669 0.91

Ca8-P vs. pH after 90 days y = 11.192 pH + 0.7887 0.94

Ca8-P vs. CaCO3 after 30 days y = 39.523 CaCO3 - 191.75 0.93






Ca10-P vs. CaCO3 after 30 days y = 11.368 CaCO3 + 226.38 0.69



BAP vs. pH after 30 days y = -23.944 pH + 194.22 0.92

Continued

101

Table page 2

Variables Linear regression equation (R2)

BAP P vs. pH after 60 days y = -23.348 pH + 189.82 0.94

BAP P vs. pH after 90 days y = -27.045 pH + 218.82 0.98

BAP vs. CaCO3 after 30 days y = -56.31 CaCO3 + 406.54 0.86



Zn vs. pH after 30 days y = -2.9279 pH + 23.465 0.94



Zn vs. CaCO3 after 30 days y = -6.7338 CaCO3 + 48.351 0.84



Fe vs. pH after 30 days y = -9.0326 pH + 74.593 0.90



Fe vs. CaCO3 after 30 days y = -19.201 CaCO3 + 140.23 0.69



Mn vs. pH after 30 days y = -4.8337 pH + 38.775 0.89



Mn vs. CaCO3 after 30 days y = -10.059 CaCO3 + 72.366 0.65



Cu vs. pH after 30 days y = -0.7361 pH + 6.0028 0.92



Cu vs. CaCO3 after 30 days y = -1.6791 CaCO3 + 12.162 0.81

Continued

102

Table page 3

Variables Linear regression equation (R2)



B vs. pH after 30 days y = -1.8146 pH + 14.674 0.94



B vs. CaCO3 after 30 days y = -4.1803 CaCO3 + 30.147 0.84



BAP: Bio-available phosphorous

103

Table 29. Interrelationship among various soil phosphorous fractions

Para meters

Ca2-P Ca8-P Ca10-P Al-P Fe-P O-P

After 30 days

Ca8-P -0.98**

Ca10-P -0.80** 0.71**

Al-P -0.82** 0.81** 0.66**

Fe-P -0.94** 0.91** 0.77** 0.72**

O-P -0.84** 0.79** 0.81** 0.82** 0.82**

BAP 0.98** -0.95** -0.81** -0.93** -0.93** -0.79**

After 60 days

Ca8-P -0.99**

Ca10-P -0.97** 0.95**

Al-P -0.91** 0.89** 0.90**

Fe-P -0.94** 0.93** 0.93** 0.86**

O-P -0.92** 0.90** 0.90** 0.90** 0.92**

BAP 0.99** -0.99** -0.97** -0.90** -0.95** -0.91**

After 90 days

Ca8-P -0.98**

Ca10-P -0.98** 0.95**

Al-P -0.98** 0.94** 0.97**

Fe-P -0.91** 0.89** 0.86** 0.88**

O-P -0.88** 0.86** 0.84** 0.92** 0.78**

BAP 0.99** -0.99** -0.97** -0.96** -0.90** -0.88**

* p ≥ F 0.05 ** p ≥ F 0.01; BAP: Bio-available phosphorous

104

correlation (Table 27). The incubated soil pH had a strong negative significant

correlation with ECe, Ca2-P, bio-available P, Zn, Fe, Mn, Cu and B, while it had

high positive significant correlation with CaCO3, Ca8-P and Ca10-P contents in the

soil. Decrease in soil pH increased the quantities of Ca2-P, bio-available P, Zn, Fe,

Mn, Cu and B linearly in the incubated soil solution which resulted increase in

ECe and at the same time the amount of CaCO3, Ca8-P and Ca10-P decreased. The

association among these variables was confirmed by straight regression lines

(Figures 5, 6, 7 and 8). The values of coefficient of determination among these

variables proved the interdependence and significance of the data (Table 28).

Garcia et al. (2007) and Yang et al. (2010) reported similar findings about the

relationship of soil pH and CaCO3 contents with the bioavailability of various

nutrients in the soil.

Electrical conductivity of the incubated soil exhibited a huge negative

significant relationship with CaCO3, Ca8-P and Ca10-P contents and an immense

positive correlation with Ca2-P, bio-available P, Zn, Fe, Mn, Cu and B quantities

in the soil. The results were conformed by the finding of Yang et al. (2010) who

also reported an increase in soil ECe with increase in the concentration of bio-

available P, sulfates and micronutrients in the soil solution.

The association of soil CaCO3 contents was found negative significant

with the concentration of Ca2-P, bio-available P, Zn, Fe, Mn, Cu and B, and

positive significant with the quantities of Ca8-P and Ca10-P in the soil. It indicated

105

that CaCO3 contents controlled the concentration of bio-available P, Ca2-P and

micronutrients in soil solution and the quantities of bio-available P, Ca2-P and

micronutrients increased when soil CaCO3 contents decreased. However, the

amount of Ca8-P and Ca10-P was more at high CaCO3 contents level in soil.

Straight regression lines among these variables confirmed their linear relationships

(Figures 10, 11, 12, 13 and 14). These findings were in line with the results

reported by Samadi (2006) and Garcia et al. (2007).

4.2.10 Interdependence among Various Soil Phosphorous Fractions

Table 29 portrayed the data concerning interrelationship of various P

fractions among themselves and with bio-available P contents in the incubated

soil. The concentration of Ca2-P had a high negative significant correlation with

Ca8-P, Ca10-P, Al-P, Fe-P and O-P and a huge positive significant relationship

with bio-available P contents in the soil as described by Samadi (2006). The

quantities of Ca8-P depicted a significant positive association with Ca10-P, Al-P,

Fe-P and O-P and a massive negative significant relationship with bio-available P

concentration in the incubated soil. The alliance of Ca10-P contents with Al-P, Fe-

P and O-P quantities was positive significant and it was negative significant with

bio-available P contents in the soil. Samavati and Hossinpur (2006) and Adhami et

al. (2007) recorded similar correlations and relationships among various inorganic

P fractions in soil.

106

y = -2.9279x + 23.465

R2 = 0.94

0.0

0.5

1.0

1.5

2.0

2.5

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Zn

(mg

kg-1

) y = -6.7338x + 48.351

R2 = 0.84

0.0

0.5

1.0

1.5

2.0

2.5

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Zn

(mg

kg-1

)

y = -2.5176x + 20.256

R2 = 0.90

0.0

0.5

1.0

1.5

2.0

2.5

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Zn

(mg

kg-1

)

y = -8.0413x + 57.621

R2 = 0.85

0.0

0.5

1.0

1.5

2.0

2.5

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Zn

(mg

kg-1

)

y = -2.5685x + 20.78

R2 = 0.85

0.0

0.5

1.0

1.5

2.0

2.5

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Zn

(mg

kg-1

)

y = -9.7005x + 69.415

R2 = 0.85

0.0

0.5

1.0

1.5

2.0

2.5

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Zn

(mg

kg-1

)

Figure 10. Zinc extractable relation with soil pH (a) and CaCO3 (b) changes


respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

107

y = -9.0326x + 74.593

R2 = 0.90

2

4

6

8

10

12

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Fe

(mg

kg-1

) y = -19.201x + 140.23

R2 = 0.69

2468

1012

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Fe

(mg

kg-1

)

y = -8.6406x + 71.759

R2 = 0.86

2

4

6

8

10

12

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Fe

(mg

kg-1

) y = -27.419x + 198.73

R2 = 0.80

2468

1012

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Fe

(mg

kg-1

)

y = -8.2356x + 68.901

R2 = 0.92

2

4

6

8

10

12

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Fe

(mg

kg-1

)

\

y = -29.431x + 213.05

R2 = 0.84

2468

1012

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Fe

(mg

kg-1

)

Figure 11. Relation of iron extractable with soil pH (a) and CaCO3 (b) changes


respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

108

y = -4.8337x + 38.775

R2 = 0.89

0

1

2

3

4

5

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Mn

(mg

kg-1

) y = -10.059x + 72.366

R2 = 0.65

012345

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Mn

(mg

kg-1

)

y = -5.1573x + 41.388

R2 = 0.89

0

1

2

3

4

5

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Mn

(mg

kg-1

) y = -16.025x + 114.77

R2 = 0.80

012345

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Mn

(mg

kg-1

)

y = -4.8721x + 39.286

R2 = 0.87

0

1

2

3

4

5

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Mn

(mg

kg-1

) y = -17.477x + 125.03

R2 = 0.80

012345

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Mn

(mg

kg-1

)

Figure 12. Manganese extractable relation with pH (a) and CaCO3 (b) changes


respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

109

y = -0.7361x + 6.0028

R2 = 0.92

0.0

0.2

0.4

0.6

0.8

1.0

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Cu

(mg

kg-1

) y = -1.6791x + 12.162

R2 = 0.81

0.00.20.40.60.81.0

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Cu

(mg

kg-1

)

y = -0.7575x + 6.186

R2 = 0.93

0.0

0.2

0.4

0.6

0.8

1.0

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Cu

(mg

kg-1

) y = -2.3978x + 17.275

R2 = 0.87

0.00.20.40.60.81.0

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Cu

(mg

kg-1

)

y = -0.7765x + 6.3731

R2 = 0.88

0.0

0.2

0.4

0.6

0.8

1.0

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

Cu

(mg

kg-1

) y = -2.8982x + 20.834

R2 = 0.87

0.00.20.40.60.81.0

6.9 7.0 7.1 7.2

CaCO3 contents (%)

Cu

(mg

kg-1

)

Figure 13. Relation of copper extractable with pH (a) and CaCO3 (b) changes during

incubation: a1 to a3 and b1 to b3 represent data from day 30, 60 and 90, respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

110

y = -1.8146x + 14.674

R2 = 0.94

0.0

0.4

0.8

1.2

1.6

2.0

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

B (

mg

kg-1

) y = -4.1803x + 30.147

R2 = 0.84

0.0

0.40.8

1.21.6

2.0

6.9 7.0 7.1 7.2

CaCO3 contents (%)

B (

mg

kg-1

)

y = -1.4927x + 12.164

R2 = 0.90

0.0

0.4

0.8

1.2

1.6

2.0

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

B (

mg

kg-1

)

y = -4.8007x + 34.55

R2 = 0.86

0.0

0.40.8

1.21.6

2.0

6.9 7.0 7.1 7.2

CaCO3 contents (%)

B (

mg

kg-1

)

y = -1.5038x + 12.288

R2 = 0.93

0.0

0.4

0.8

1.2

1.6

2.0

7.0 7.2 7.4 7.6 7.8 8.0

Soil pH

B (

mg

kg-1

) y = -5.4995x + 39.494

R2 = 0.88

0.00.4

0.81.2

1.62.0

6.9 7.0 7.1 7.2

CaCO3 contents (%)

B (

mg

kg-1

)

Figure 14. Boron extractable relation with pH (a) and CaCO3 (b) changes during


respectively

(a1)

(a3)

(a2)

(b3)

(b2)

(b1)

111

4.3 SULFUR OXIDIZING BACTERIA APPLICATION ON PLANT

GROWTH AND P UPTAKE

4.3.1 Basic Soil Analyses of Rice and Wheat Field

The field soil was nonsaline, alkaline and calcareous in nature (Table 30).

Total P concentration in the soil was 690.45 mg kg-1, while Olsen P (4.6 mg kg-1)

was noted within deficient limit (< 5 mg kg-1) as described by Ahmad et al.

(2003). Total S and water soluble S in soil was determined as 202.7 mg kg-1 and

9.5 mg kg-1, respectively in the experimental soil. Quantity of water soluble S was

< 10 mg kg-1 which was deficient for plant growth.

The concentration of total S and water soluble S in the soil was in similar

range as reported by Srinivasarao et al. (2004) and Mohammed and Adamu

(2009). Quantities of Zn, Fe, Mn, Cu and B were recorded as 0.32, 3.33, 0.67,

0.19 and 0.27 mg kg-1, respectively. Zinc, Mn and B contents in the soil were

deficient while Fe and Cu were adequate. These results were in harmony with the

findings obtained by Zia et al. (2006) and Niaz et al. (2007) regarding

micronutrients status of Pakistani soils.

4.3.2 Treatment Effect on Bio-available Phosphorous Contents at Rice

Harvest

The highest values of plant available P in soil (14.2 mg kg-1 with net

increase of 9.6 mg kg-1) were recorded with S IW16 (Thiobacillus strain IW16

112

Table 30. Basic soil analyses before rice-wheat experiment


ECe dS m-1 1.1

pH - 8.1

O.M % 0.4

CaCO3 % 7.0


Available phosphorous mg kg-1 4.6


Water soluble sulfur mg kg-1 9.5

Zinc mg kg-1 0.32

Iron mg kg-1 3.33

Mangnese mg kg-1 0.67

Copper mg kg-1 0.19

Boron mg kg-1 0.27

Clay % 18.2

Sand % 70.9

Silt % 11.0


113

Table 31. Soil bio-available phosphorous contents at rice harvest

Treatments

Bio-available P

Net increase / decrease over the initial value

---------------------- mg kg-1-----------------------

Control 4.1 k -0.5

IW16 5.7 f 1.1

SW2 4.7 i 0.1

P1 4.5 j -0.1

P1 IW16 5.9 e 1.3

P1 SW2 4.9 h 0.3

P2 4.6 ij -

P2 IW16 6.8 d 2.2

P2 SW2 5.7 f 1.1

S 5.6 f 1.0

S IW16 14.2 a 9.6

S SW2 11.5 c 6.9

P1S 5.0 g 0.4

P1S IW16 13.7 b 9.1

P1S SW2 11.5 c 6.9

LSD 0.14


114

with 100 kg S ha-1) and the lowest values of plant available P in soil (4.1 mg kg-1

with net decrease of 0.5 mg kg-1) were noted in control treatment (Table 31).

Concentration of bio-available P decreased in control and in P1 treatment plots (45

kg P2O5 ha-1) due to continuous plant uptake without solubilization and no / less

addition of P in soil. Intensity of P solubilization in soil was less in treatments

where Thiobacilli and S were used separately than those where they were used

collectively. The enhancement and bioavailability of plant available P in soil had a

positive significant correlation with bacterial S oxidation mechanism occurred

under the combined effect of Thiobacilli and S. These findings were conformed by

the findings of Stamford et al. (2003) and Hassan et al. (2010) who also recorded

P solubilization under the interactive effect of S and Thiobacilli.

4.3.3 Treatment Effect on Growth and Yield Attributes of Rice

Table 32 represents the data pertaining to plant height, panicle length,

number of tillers plant-1, grains panicle -1 and 1000 grains weight under treatments

effect in rice crop. Maximum plant height (122.0 cm), panicle length (29.9 cm),

number of tillers Plant-1 (21.2), number of grains panicle-1 (162.4) were

recorded under treatment P1S IW16 (Thiobacillus strain IW16, 100 kg S ha-1 and

45 kg P2O5 ha-1). While maximum 1000 grains weight (32.5 g) was noted with

treatment S IW16 (Thiobacillus strain IW16 plus 100 kg S ha-1). Minimum plant

height (101.3 cm), panicle length (21.2 cm), number of tillers Plant-1(9.6), number

of grains panicle-1 (98.4) and 1000 grains weight (19.6 g) were recorded in control

plots. Treatments S IW16 and P1S IW16 were found statistically identical. Sulfur

115

Table 32. Treatment effect on growth parameters of rice

Treatments

Plant height Panicle length

Tillers plant -1

Grains panicle -1

1000 grain weight

----------- cm ----------- -----------No.--------- --- g ---

Control 101.3 h 21.2 h 9.6 h 98.4 l 19.6 i

IW16 108.6 de 25.5 de 14.6 de 132.6 e 25.2 ef

SW2 106.3 fg 25.0 f 13.0 g 119.5 j 23.4 g

P1 105.2 g 24.5 g 12.8 g 115.2 k 22.9 h

P1 IW16 110.4 d 25.9 cd 14.9 d 136.5 d 25.6 e

P1 SW2 107.8 ef 25.1 ef 13.2 g 125.8 g 23.6 g

P2 107.5 ef 25.3 ef 14.4 e 123.5 h 24.8 f

P2 IW16 112.7 c 26.1 c 15.8 c 139.6 c 27.0 d

P2 SW2 109.9 d 25.3 ef 14.4 e 130.5 f 25.1 ef

S 106.5 fg 25.1 ef 13.8 f 118.7 j 24.9 f

S IW16 121.9 a 29.7 a 21.2 a 161.3 a 32.5 a

S SW2 117.3 b 28.1 b 18.5 b 153.9 b 30.0 b

P1S 108.8 de 25.5 def 14.5 e 121.6 i 25.2 ef

P1S IW16 122.0 a 29.9 a 21.2 a 162.4 a 32.1 a

P1S SW2 117.6 b 28.2 b 18.6 b 154.5 b 29.2 c

LSD 1.8 0.5 0.4 1.8 0.5


116

Table 33. Treatment effect on yield attributes of rice

Treatments Paddy yield Straw yield Biological yield

Harvest index

-------------------- Mg ha-1--------------- --- % ---

Control 2.98 j 5.03 g 8.01 h 37.20 i

IW16 6.31 def 5.97 c 12.28 e 51.38 efg

SW2 5.56 hi 5.52 de 11.08 f 50.18 g

P1 5.22 i 5.14 fg 10.36 g 50.39 g

P1 IW16 6.51 de 7.03 b 13.54 d 48.08 h

P1 SW2 5.72 gh 5.55 de 11.27 f 50.75 fg

P2 6.24 def 5.25 efg 11.49 f 54.31 a-d

P2 IW16 7.03 c 7.11 ab 14.14 c 49.72 gh

P2 SW2 6.57 d 5.71 cd 12.28 e 53.50 bcd

S 5.98 fg 5.40 def 11.38 f 52.55 def

S IW16 9.42 a 7.48 a 16.90 a 55.74 a

S SW2 8.58 b 7.16 ab 15.74 b 54.51 a-d

P1S 6.12 ef 5.45 def 11.57 f 52.90 cde

P1S IW16 9.16 a 7.41 a 16.57 a 55.28 ab

P1S SW2 8.63 b 7.15 ab 15.78 b 54.69 abc

LSD 0.37 0.33 0.51 1.88


117

oxidation treatments with or without P significantly increased crop growth and

yield parameters by enhancing bioavailability of P in soil. The results were

supported by Alam et al. (2009) who recorded an increase in number of tillers

Plant-1, panicle length, number of grains panicle-1, number of grains panicle-1 and

1000 grains weight in rice crop by increasing the concentration of plant available

P in soil. Khan et al. (2007) also reported significant effect of increased quantity

of plant available P in soil on plant length and panicle length in rice crop.

Paddy, straw and biological yields and harvest index recorded at harvest

are given in Table 33. The highest values of paddy (9.42 Mg ha-1), straw (7.48 Mg

ha-1) and biological yields (16.90 Mg ha-1), and harvest index (55.74 %) were

recorded with S IW16 (Thiobacillus strain IW16 plus 100 kg S ha-1). The lowest

values of paddy (2.98 Mg ha-1), straw (5.03 Mg ha-1) and biological yields (8.01

Mg ha-1), and harvest index (37.20 %) were determined in control. Yield attributes

of rice increased in treatment plots where the amount of plant available P

increased as a result of bacterial S oxidation under the effect of Thiobacilli or S

and the increase was more acute when Thiobacilli and S were used together. Khan

et al. (2007) and Rahman et al. (2007) reported that the yield of rice crop

increased with the increase in P and S quantities in soil. Findings of Afzal and

Bano (2008) were also quite in line with these results. Panhwar et al. (2011) also

concluded similar results and found that yield of rice crop was significantly

correlated with the extent of P solubilization in soil occurred under the influence

of phosphorous solubilizing bacteria.

118

Table 34. Phosphorous uptake by rice

Treatments

P concentration P uptake

Paddy Straw Paddy Straw Total

------------ % ---------- ----------------- kg ha-1-----------------

Control 0.081 l 0.014 n 2.41 k 0.70 l 3.11 l

IW16 0.172 g 0.039 k 10.85 f 2.33 hi 13.18 g

SW2 0.159 j 0.030 l 8.84 i 1.66 j 10.50 j

P1 0.151 k 0.021 m 7.88 j 1.08 k 8.96 k

P1 IW16 0.179 f 0.062 g 11.65 e 4.36 g 16.01 f

P1 SW2 0.162 i 0.043 j 9.27 hi 2.39 h 11.66 i

P2 0.158 j 0.045 i 9.86 gh 2.36 hi 12.22 hi

P2 IW16 0.184 e 0.095 e 12.94 d 6.75 e 19.69 e

P2 SW2 0.173 g 0.087 f 11.37 ef 4.97 f 16.34 f

S 0.161 i 0.039 k 9.63 gh 2.11 i 11.74 i

S IW16 0.272 b 0.129 a 25.62 a 9.65 a 35.27 a

S SW2 0.241 d 0.112 d 20.68 c 8.02 d 28.70 d

P1S 0.164 h 0.047 h 10.04 g 2.56 h 12.60 gh

P1S IW16 0.278 a 0.123 b 25.46 a 9.11 b 34.57 b

P1S SW2 0.248 c 0.118 c 21.40 b 8.44 c 29.84 c

LSD 0.014 0.002 0.66 0.25 0.70


119

4.3.4 Phosphorous Uptake by Rice

Quantities of P concentration and P uptake in paddy had the highest values

(0.278 % and 25.62 kg ha-1, respectively) with P1S IW16 where Thiobacillus strain

IW16 was inoculated with 100 kg S ha-1 and 45 kg P2O5 ha-1 as DAP (Table 34).

Maximum P concentration and P uptake in straw (0.129 % and 9.65 kg ha-1,

respectively) was noted in treatment S IW16 which contained Thiobacillus strain

IW16 with 100 kg S ha-1. Control plots had the lowest values of P concentration

and P uptake in paddy (0.081 % and 2.41 kg ha-1, respectively) and straw (0.014 %

and 0.70 kg ha-1, respectively).

Phosphorous concentration and P uptake in paddy and straw was

significantly correlated with the amount of plant available P in soil. Therefore, the

treatments which solubilized and enhanced high quantity of P in soil through S

oxidation by Thiobacilli also had high amount of P in paddy and straw. Slaton et

al. (2001) and Singh et al. (2002) reported similar results and concluded a positive

significant correlation between the quantity of P in rice plants with the

concentration of plant available P in soil.

4.3.5 Correlation among Various Soil and Rice Variables

Relationship between various soil and plant variables in rice crop was

studied through determination of correlation coefficient, linear regression

equations and coefficient of determinations (Tables 35 and 36). Quantities of

different rice crop variables (total P uptake, paddy, straw and biological yields and

120

Table 35. Correlation among various rice variables

Parameters Bio-available P

Total P uptake

Paddy yield Straw yield

Biological yield

Total P uptake 0.97**

Paddy yield 0.91** 0.97**

Straw yield 0.86** 0.90** 0.86**

Biological yield

0.92** 0.98** 0.98** 0.94**

Harvest index 0.55* 0.66** 0.80** 0.42 0.69**

* p ≥ F 0.05 ** p ≥ F 0.01

121

Table 36. Linear regression analyses among soil and rice variables


(R2)

TPU vs. BAP y = 2.7002BAP - 1.8719 0.95

1000 GW vs. BAP y = 0.9392 BAP + 19.284 0.89

PY vs. BAP y = 0.4331 BAP + 3.5415 0.82

SY vs. BAP y = 0.2245 BAP + 4.5361 0.73

BY vs. BAP y = 0.6576 BAP + 8.0776 0.84

PY vs. TPU y = 0.1672 TPU + 3.7213 0.94

SY vs. TPU y = 0.0853 TPU + 4.6535 0.81

BY vs. TPU y = 0.2525 TPU + 8.3748 0.95

HI vs. TPU y = 0.3042 TPU + 46.05 0.44

BAP: Bio-available phosphorous, TPU: Total phosphorous uptake, PY: Paddy yield, SY: Straw yield, BY: Biological yield, HI: Harvest index

122

y = 2.7002x - 1.8719

R2 = 0.95

0

8

16

24

32

40

0 4 8 12 16 20

Bioavailable P (mg kg-1)

Tot

al P

upt

ake

(kg

ha-1

)

y = 0.4331x + 3.5415

R2 = 0.82

0

2

4

6

8

10

0 4 8 12 16 20


Pad

dy y

ield

(M

g ha

-1)

y = 0.2245x + 4.5361

R2 = 0.73

0

2

4

6

8

10

0 4 8 12 16 20


Str

aw y

ield

(M

g ha

-1)

y = 0.6576x + 8.0776

R2 = 0.84

0

4

8

12

16

20

0 4 8 12 16 20


Bio

logi

cal y

ield

(M

g ha-1

)

Figure 15. Soil bio-available P relation with different rice yield attributes

123

y = 0.1672x + 3.7213

R2 = 0.94

0

2

4

6

8

10

0 8 16 24 32 40

Total P uptake (kg ha-1)

Pad

dy y

ield

(M

g ha

-1)

y = 0.0853x + 4.6535

R2 = 0.81

0

2

4

6

8

10

0 8 16 24 32 40


Str

aw y

ield

(M

g ha

-1)

y = 0.2525x + 8.3748

R2 = 0.95

0

4

8

12

16

20

0 8 16 24 32 40


Bio

logi

cal y

ield

(M

g ha-1

)

y = 0.3042x + 46.05

R2 = 0.44

35

40

45

50

55

60

0 8 16 24 32 40


Har

vest

inde

x (%

)

Figure 16. Phosphorous uptake relation with different rice yield attributes

124

harvest index) increased in a linear fashion with increase in the concentration of

bio-available P (Figure 15).

Similarly, total P uptake had a strong positive significant affiliation with all

yield attributes of rice crop (paddy, straw and biological yields, and harvest

index). High uptake of P by rice crop plants increased paddy, straw and biological

yields, and harvest index in a straight line (Figure 16). Afzal and Bano (2008) and

Panhwar et al. (2011) reported similar relationship between different crop

variables and amount of plant available P in the soil solution.

4.3.6 Treatment Effect on Bio-available Phosphorous Contents at Wheat

Harvest

The highest quantity of soil bio-available P (16.5 mg kg-1 with net increase

of 11.9 mg kg-1) was noted with S IW16 (Thiobacillus strain IW16 with 100 kg S

ha-1), while the lowest values of bio-available P in soil (3.9 mg kg-1 with net

decrease of 0.7 mg kg-1) were recorded with control (Table 37). Amount of soil

bio-available P decreased in treatment P1 (45 kg P2O5 ha-1) due to less addition

and more plant uptake. Similarly, decrease in the concentration of soil bio-

available P was more in control plots due to P uptake by plants in the absence of P

solubilization / addition. Aria et al. (2010) and Oh et al. (2010) also reported an

increase in the amount of plant available P through P solubilization and

enhancement in soil by the combined effect of Thiobacilli and S.

125

Table 37. Soil bio-available phosphorous contents at wheat harvest

Treatments Bio-available P

Net increase / decrease over the initial value

---------------------- mg kg-1---------------------

Control 3.9 k -0.7

IW16 6.8 d 2.2

SW2 5.1 hi 0.5

P1 4.4 j -0.2

P1 IW16 7.0 d 2.4

P1 SW2 5.3 gh 0.7

P2 4.8 ij 0.2

P2 IW16 7.9 c 3.3

P2 SW2 6.3 e 1.7

S 5.8 f 1.2

S IW16 16.5 a 11.9

S SW2 13.9 b 9.3

P1S 5.6 fg 1.0

P1S IW16 16.2 a 11.6

P1S SW2 13.7 b 9.1

LSD 0.36


126

Table 38. Treatment effect on growth parameters of wheat

Treatments

Plant height Spike length

Tillers plant -1

Grains Spike -1

1000 grain weight

---------- cm --------- -----------No.--------- --- g---

Control 90.2 g 9.7 j 3.3 h 26.7 n 40.9 l

IW16 94.5 de 12.3 e 4.6 e 29.5 k 43.2 i

SW2 93.0 ef 11.5 h 4.2 f 28.5 l 42.8 j

P1 92.1 f 10.9 i 3.8 g 27.9 m 42.0 k

P1 IW16 95.6 cd 12.9 d 4.8 d 31.7 f 44.5 f

P1 SW2 94.2 de 11.9 fg 4.3 f 30.7 j 43.8 h

P2 94.1 cd 11.8 g 4.3 f 30.8 ij 43.7 h

P2 IW16 96.8 c 13.8 c 5.1 c 32.6 e 44.9 e

P2 SW2 95.3 cd 12.4 e 4.7 d 31.3 g 44.2 g

S 94.1 de 11.9 g 4.5 e 30.9 hi 43.7 h

S IW16 104.9 a 16.3 a 8.9 a 42.9 b 50.3 a

S SW2 102.2 b 15.2 b 7.7 b 39.4 c 48.5 c

P1S 94.3 de 12.1 f 4.6 e 31.1 h 43.8 h

P1S IW16 105.3 a 16.2 a 9.0 a 43.2 a 49.6 b

P1S SW2 102.6 b 15.3 b 7.8 b 38.9 d 47.6 d

LSD 1.5 0.2 0.1 0.2 0.3


127

Table 39. Treatment effect on Yield attributes of wheat

Treatments Grain yield Straw yield Biological yield

Harvest index

------------------- Mg ha-1----------------- --- % ---

Control 0.89 l 1.15 j 2.04 l 43.63 g

IW16 2.78 i 3.04 h 5.82 i 47.77 def

SW2 2.63 j 2.96 h 5.59 j 47.05 ef

P1 2.39 k 2.73 i 5.12 k 46.68 f

P1 IW16 3.16 g 3.36 f 6.52 g 48.47 cd

P1 SW2 2.99 h 3.25 g 6.24 h 47.92 de

P2 3.07 gh 3.38 f 6.45 g 47.60 def

P2 IW16 3.54 e 3.63 e 7.17 e 49.37 bc

P2 SW2 3.34 f 3.53 e 6.87 f 48.62 cd

S 2.98 h 3.21 g 6.19 h 48.14 cde

S IW16 5.34 a 5.17 a 10.51 a 50.81 a

S SW2 4.82 c 4.76 c 9.58 c 50.31 ab

P1S 3.11 g 3.30 fg 6.41 g 48.52 cd

P1S IW16 4.97 b 4.90 b 9.87 b 50.35 ab

P1S SW2 4.60 d 4.55 d 9.15 d 50.27 ab

LSD 0.11 0.11 0.15 1.11


128

4.3.7 Treatment Effect on Growth and Yield Attributes of Wheat

Data stand for plant height, spike length, number of tillers plant-1, number

of grains spike-1 and 1000 grain weight are given in Table 38. The highest plant

height (105.3 cm), number of tillers plant-1 (9.0) and grains spike -1 (43.2) were

recorded in treatment P1S IW16 (Thiobacillus strain IW16, 100 kg S ha-1 and 45

kg P2O5 ha-1), while the highest spike length (16.3 cm) and 1000 grains weight

(50.3 g) were noted in S IW16 (Thiobacillus strain IW16 plus 100 kg S ha-1). Both

the treatments S IW16 and P1S IW16 were found statistically similar in growth

parameters of wheat crop. The lowest values of plant height (90.2 cm), spike

length (9.7 cm), number of tillers plant-1 (3.3), number of grains spike -1 (26.7) and

1000 grains weight (40.9 g) were recorded in control treatment. The treatments

containing high amount of bio-available P as a consequent of bacterial

solubilization and enhancement had a positive significant effect on plant height,

spike length, number of tillers plant-1, number of grains spike -1 and 1000 grains

weight in wheat crop. Alam et al. (2003) and Hussain et al. (2008) reported

similar findings of increase in plant height, spike length and number of tillers

plant-1 in wheat crop by increasing bio-available concentration of P in soil.

Hussain et al. (2004) and Kaleem et al. (2009) recorded an increase in grains

spike-1 and 1000 grains weight in wheat crop under the effect of high

concentration of bio-available P in soil.

Maximum values of grain (5.34 Mg ha-1), straw (5.17 Mg ha-1) and

biological yields (10.51 Mg ha-1), and harvest index (50.81 %) were recorded with

129

S IW16, while minimum values of grain (0.89 Mg ha-1), straw (1.15 Mg ha-1) and

biological yields (2.04 Mg ha-1), and harvest index (43.63 %) were noted in

control plots (Table 39). High significant difference with control in the yield

attributes of wheat (grain, straw and biological yields, and harvest index) was

observed in S oxidation treatments. Positive significant relationship of P

concentration in soil solution with grain, straw and biological yields and harvest

index of wheat crop was recorded. These results were supported by the findings of

Yadav et al. (2011) who reported an increase in wheat yield by enhancing soil P

bioavailability under the influence of phosphorous solubilizing bacteria. Rahman

et al. (2007) also reported that high dose of S application significantly increased

both grain and straw yields of different crops.

4.3.8 Phosphorous Upatake by Wheat

Maximum concentration of P in wheat grain (0.426 %) was recorded with

P1S IW16 (Thiobacillus strain IW16, 100 kg S ha-1 plus 45 kg P2O5 ha-1) and the

highest value of P concentration in wheat straw (0.171 %) was noted with S IW16

(Table 40). The highest P uptake by wheat grain, wheat straw and subsequently

the total P uptake (22.05, 8.84 and 30.89 kg ha-1, respectively) was determined in

treatment S IW16, whereas the lowest quantity of P concentration in wheat grain

and wheat straw was determined as 0.121 and 0.016 %, respectively and the

lowest quantity of P uptake in wheat grain and wheat straw was recorded as 1.08

and 0.18 kg ha-1, respectively in control plots. The treatments which solubilized

and enhanced high quantity of P in soil contained high amount of P in wheat grain

130

Table 40. Phosphorous uptake by wheat

Treatments


Grain Straw Grain Straw Total

----------- % ---------- ----------------- kg ha-1 ----------------

Control 0.121 n 0.016 o 1.08 m 0.18 n 1.26 n

IW16 0.214 k 0.045 l 5.95 j 1.37 k 7.32 k

SW2 0.202 l 0.033 m 5.31 k 0.98 l 6.29 l

P1 0.184 m 0.029 n 4.40 l 0.79 m 5.19 m

P1 IW16 0.248 g 0.072 g 7.84 g 2.42 g 10.26 g

P1 SW2 0.233 hi 0.057 i 6.97 i 1.85 hi 8.82 i

P2 0.231 i 0.052 j 7.09 hi 1.76 i 8.85 i

P2 IW16 0.286 e 0.113 e 10.12 e 4.10 e 14.22 e

P2 SW2 0.268 f 0.098 f 8.95 f 3.46 f 12.41 f

S 0.228 j 0.049 k 6.79 i 1.57 j 8.36 j

S IW16 0.413 b 0.171 a 22.05 a 8.84 a 30.89 a

S SW2 0.367 d 0.158 c 17.69 c 7.52 c 25.21 c

P1S 0.236 h 0.059 h 7.34 h 1.95 h 9.29 h

P1S IW16 0.426 a 0.169 b 21.17 b 8.28 b 29.45 b

P1S SW2 0.374 c 0.152 d 17.20 d 6.92 d 24.12 d

LSD 0.003 0.002 0.299 0.118 0.335


131

and wheat straw. Therefore, a positive significant correlation was found between

soil available P and P contents in wheat plants. The results were in conformity

with those of Patidar and Mali (2001) and Alam et al. (2003) who reported an

increase in P concentration in wheat grain and wheat straw with the increase in

plant available P in soil.

4.3.9 Correlation among Various Soil and Wheat Variables

Interrelationship and interdependence data about various soil and wheat

crop variables are enclosed in Table 41 and 42, respectively. High concentration

of bio-available P in soil solution raised the quantities of total P uptake, wheat

grain, straw and biological yields linearly and with increase in the amount of total

P uptake, wheat grain, straw and biological yields and harvest index also increased

in a linear fashion (huge positive significant correlation).

Interdependence of various variables was confirmed by the values of

linear regression equations and coefficient of determinations. Figures 17 and 18

depicted that bio-available P contents in soil, P uptake by wheat crop and yield

attributes of wheat crop are inter-dependable and interrelated. These results were

in line with those of Hussain et al. (2008) and Yadav et al. (2011) who also

recorded similar kind of relationships between the amount of bio-available P in

soil and different yield crop variables.

132

Table 41. Correlation among various wheat variables

Parameters Bio-available P

Total P uptake

Paddy yield Straw yield

Biological yield


Paddy yield 0.84** 0.96**

Straw yield 0.83** 0.94** 0.99**

Biological yield

0.84** 0.95** 0.99** 0.99**

Harvest index 0.86** 0.88** 0.97** 0.97** 0.97**

* p ≥ F 0.05 ** p ≥ F 0.01

133

Table 42. Linear regression analyses among soil and wheat variables


(R2)

TPU vs. BAP y = 2.0546 BAP - 3.3817 0.96

1000 GW vs. BAP y = 0.611 BAP + 39.881 0.95

WGY vs. BAP y = 0.2365 BAP + 1.4353 0.84

WSY vs. BAP y = 0.2009 BAP + 1.8812 0.78

BY vs. BAP y = 0.4373 BAP + 3.3165 0.81

WGY vs. TPU y = 0.1188 TPU + 1.7747 0.92

WSY vs. TPU y = 0.1018 TPU + 2.1573 0.88

BY vs. TPU y = 0.2206 TPU + 3.932 0.91

HI vs. TPU y = 0.1716 TPU + 46.058 0.77

BAP: Bio-available phosphorous, TPU: Total phosphorous uptake, WGY: Wheat grain yield, WSY: Wheat straw yield, BY: Biological yield, HI: Harvest index

134

y = 2.0546x - 3.3817

R2 = 0.96

0

8

16

24

32

40

0 4 8 12 16 20


Tot

al P

upt

ake

(kg

ha-1

)y = 0.2365x + 1.4353

R2 = 0.84

0

2

3

5

6

8

0 4 8 12 16 20


Whe

at g

rain

yie

ld (

Mg

ha-1)

y = 0.2009x + 1.8812

R2 = 0.78

0

2

3

5

6

8

0 4 8 12 16 20


Whe

at s

traw

yie

ld (

Mg

ha-1) y = 0.4373x + 3.3165

R2 = 0.81

0

3

6

9

12

15

0 4 8 12 16 20


Bio

logi

cal y

ield

(M

g ha-1

)

Figure 17. Soil bio-available P relation with different wheat yield attributes

135

y = 0.1188x + 1.7747

R2 = 0.92

0

2

3

5

6

8

0 8 16 24 32 40


Whe

at g

rain

yie

ld (

Mg

ha-1)

y = 0.1018x + 2.1573

R2 = 0.88

0

2

3

5

6

8

0 8 16 24 32 40


Whe

at s

traw

yie

ld (

Mg

ha-1)

y = 0.2206x + 3.932

R2 = 0.91

0

3

6

9

12

15

0 8 16 24 32 40


Bio

logi

cal y

ield

(M

g ha-1

)

y = 0.1716x + 46.058

R2 = 0.77

40

43

46

49

52

55

0 8 16 24 32 40


Har

vest

inde

x (%

)

Figure 18. Phosphorous uptake relation with different wheat yield attributes

136

S IW16 Rice crop Control

S IW16 Wheat crop Control

Plate 2. Response of rice and wheat to Thiobacillus

137

4.3.10 Basic Soil Analyses for Maize Field

The experimental soil was nonsaline, alkaline and calcareous in nature

with ECe 1.17 dS m-1, pH 8.0 and CaCO3 contents 7.18 % (Table 43). These

characteristics represented most of the soils present in semi arid and arid regions

(Bryan and Jason, 2005). Quantity of total P in the soil was determined as 678.79

mg kg-1, whereas the amount of plant available P was recorded within deficient

limit (< 5 mg kg-1) like most of the Pakistani soils (Rehman et al., 2000; Solangi

et al., 2006). Concentration of total soil S and water soluble S was estimated as

207.34 and 9.26 mg kg-1, respectively.

Quantity of water soluble S came under deficient limit (< 10 mg kg-1)

required for plant growth (Ahmed et al., 1994). Concentration of Zn, Fe, Mn, Cu

and B were recorded as 0.34, 3.46, 0.69, 0.17 and 0.29 mg kg-1, respectively.

Most of the micronutrients like Zn, Mn and B in the experimental soil were

deficient (<1.0, <1.0 and <0.5 mg kg-1, respectively) for plant growth (Ryan et al.,

2001). Moreover, the results reported by Zia et al. (2006) about the micronutrient

status of Pakistani soils were quite similar with the micronutrient quantities

determined in the experimental soil.

4.3.11 Treatment Effect on Soil Bio-available Phosphorous Contents at

Maize Harvest

After spring maize crop harvest maximum significant increase in the

amount of plant available P was recorded as 14.8 mg kg-1 (net increase of 10.3 mg

138

Table 43. Basic soil analyses before spring-autumn maize experiment


ECe dS m-1 1.2

pH - 8.0

O.M % 0.4

CaCO3 % 7.2


Available p mg kg-1 4.5


Water solubke sulfur mg kg-1 9.3

Zinc mg kg-1 0.34

Iron mg kg-1 3.46

Manganese mg kg-1 0.69

Copper mg kg-1 0.17

Boron mg kg-1 0.29

Clay % 18.9

Sand % 71.6

Silt % 9.5


139

Table 44. Soil bio-available phosphorous contents at maize harvest

Treatments

Bio-available P Net increase / decrease over the

initial value

Spring Autumn Spring Autumn

-------------------------------- mg kg-1--------------------------

Control 3.9 l 3.8 k -0.6 -0.7

IW16 5.8 g 6.1 f 1.3 1.6

SW2 4.9 i 5.3 h 0.4 0.8

P1 4.4 k 4.2 j -0.2 -0.3

P1 IW16 6.5 e 6.9 d 2.0 2.4

P1 SW2 5.1 h 5.8 g 0.6 1.3

P2 4.6 j 4.6 i 0.1 0.1

P2 IW16 7.4 d 7.56c 2.9 3.1

P2 SW2 6.2 f 6.4 e 1.7 1.9

S 5.3 h 5.4 h 0.8 0.9

S IW16 14.8 a 17.2 a 10.3 12.7

S SW2 12.5 c 14.2 b 8.0 9.7

P1S 6.1 f 6.1 f 1.6 1.6

P1S IW16 14.7 a 17.3 a 10.2 12.8

P1S SW2 12.7 b 14.2 b 8.2 9.7

LSD 0.2 0.2


140

kg-1) with S IW16 where Thiobacillus strain IW16 was inoculated with 100 kg S

ha-1 and minimum values of plant available P was noted as 3.9 mg kg-1 (net

decrease of 0.6 mg kg-1) in control. Whereas, after autumn maize crop harvest the

highest increase in the amount of soil bio-available P was determined with

treatment P1S IW16 (Thiobacillus strain IW16 and 100 kg S ha-1 with 45 kg P2O5

ha-1) with values of 17.2 mg kg-1 (net increase of 12.8 mg kg-1) and minimum

concentration of soil bio-available P was determined as 3.8 mg kg-1 (net decrease

of 0.7 mg kg-1) in control treatment (Table 44). Treatment P1S IW16 was found

statistically at par with S IW16. Concentration of bio-available P in soil decreased

in control treatment due to continuous plant uptake without addition /

solubilization. Similarly, the quantity of plant available P decreased slightly in

treatments P1 (45 kg P2O5 ha-1) due to less addition and more plant uptake.

The mechanism of P solubilzation and enhancement by bacterial S

oxidation continued in autumn maize crop like spring maize crop and P

availability status of the soil increased. Phosphorous solubilization and

enhancement in soil depended on Thiobacilli spp. along with S because these two

factors are important for bacterial S oxidation as described by Besharati et al.

2007 and Hassan et al. 2010. Both these factors significantly increased plant

available P contents in soil independently (less quantity) and collectively (high

quantity). The process of P solubilization and enhancement was also observed by

Zhou et al. (2002) and Jaggi et al. (2005) as a consequent of joint effect of

Thiobacillus spp. and S.

141

Table 45. Treatment effect on growth parameters of maize

Treatments Cobs plant-1 Grains plant-1 1000 grains

weight

Spring Autumn Spring Autumn Spring Autumn

----------------------- No.------------------------ ------------ g -----------

Control 0.85 g 0.81 f 255.3 m 243.3 l 185.6 o 183.8 k

IW16 1.15 c-e 1.19 c 394.6 g 410.5 g 251.8 i 258.9 f

SW2 1.08 ef 1.14 cd 373.7 k 382.6 i 229.7 n 237.5 i

P1 1.05 f 1.01 e 370.7 l 367.4 k 230.7 m 226.2 j

P1 IW16 1.18 b-d 1.20 c 402.8 e 417.7 f 259.6 f 266.6 d

P1 SW2 1.11 c-f 1.15 cd 377.4 j 393.8 h 246.4 l 251.6 g

P2 1.10 d-f 1.08 de 375.8 j 372.6 j 249.3 j 246.4 h

P2 IW16 1.19 bc 1.24 bc 421.7 d 455.3 e 267.3 e 275.2 c

P2 SW2 1.13 c-f 1.22 bc 398.5 f 419.4 f 255.6 h 262.8 e

S 1.11 c-f 1.14 cd 379.2 i 392.8 h 247.8 k 253.3 g

S IW16 1.29 a 1.35 a 579.7 b 607.3 b 310.5 b 319.7 a

S SW2 1.24 ab 1.31 ab 542.9 c 578.7 c 291.3 d 298.8 b

P1S 1.13 c-f 1.16 cd 389.6 h 410.7 g 256.5 g 261.4 e

P1S IW16 1.30 a 1.36 a 584.3 a 611.9 a 313.2 a 319.4 a

P1S SW2 1.24 ab 1.32 ab 544.6 c 576.5 d 292.6 c 298.3 b

LSD 0.08 0.09 1.8 2.1 0.9 1.7


142

Table 46. Treatment effect on yield attributes of maize

Treatments Grain yield Stalk yield Biological yield

Spring Autumn Spring Autumn Spring Autumn

------------------------------------ Mg ha-1----------------------------------

Control 1.71 i 1.59 j 6.23 h 6.15 g 7.94 i 7.74 i

IW16 4.50 de 4.64 e 10.0 3ef 10.41 d 14.53 ef 15.05 e

SW2 4.36 g 4.40 g 9.93 fg 9.95 ef 14.29 gh 14.35 fg

P1 4.13 h 4.05 i 9.98 e-g 9.92 ef 14.11 h 13.97 h

P1 IW16 4.60 d 4.66 e 10.20 d 10.44 d 14.80 d 15.10 e

P1 SW2 4.38 fg 4.43 g 9.87 g 9.98 ef 14.25 gh 14.41 f

P2 4.37 g 4.27 h 9.94 e-g 9.82 f 14.31 f-h 14.09 gh

P2 IW16 4.74 c 5.02 d 10.41 c 11.14 c 15.15 c 16.16 d

P2 SW2 4.49 d-f 4.61 ef 10.06 de 10.35 d 14.55 e 14.96 e

S 4.43 e-g 4.49 g 10.00 d-g 10.04 e 14.43 e-g 14.53 f

S IW16 6.78 a 7.24 a 14.00 a 14.25 a 20.78 a 21.49 a

S SW2 6.37 b 6.76 c 13.69 b 13.84 b 20.06 b 20.60 c

P1S 4.46 e-g 4.51 fg 10.07 de 10.07 e 14.53 ef 14.58 f

P1S IW16 6.83 a 7.06 b 14.06 a 14.1 a 20.89 a 21.16 b

P1S SW2 6.42 b 6.79 c 13.80 b 13.87 b 20.22 b 20.66 c

LSD 0.11 0.11 0.14 0.18 0.21 0.27


143

4.3.12 Treatment Effect on Growth Parameters of Maize

Table 45 portrays the data regarding number of cobs plant-1, number of

grains plant-1 and 1000 grains weight in both spring and autumn maize crops at

harvest. In spring maize crop maximum number of cobs plant-1 (1.30) and 1000

grains weight (313.2 g) were noted in treatment P1S IW16 where Thiobacillus

strain IW16 was inoculated along with 100 kg S ha-1 and 45 kg P2O5 ha-1 DAP.

Minimum number of cobs plant-1 (0.85), number of grains plant-1 (255.3) and 1000

grains weight (185.6 g) were recorded in control plots. In autumn maize crop

the highest significant increase in number of cobs plant-1 (1.36) and number of

grains plant-1 (611.9) were recorded with P1S IW16 (Thiobacillus strain IW16 with

100 kg S ha-1 and 45 kg P2O5 ha-1) and maximum 1000 grains weight (313.2 g)

was recorded with S IW16 (Thiobacillus strain IW16 plus 100 kg S ha-1).

However, the lowest values of number of cobs plant-1 (0.81), number of grains

plant-1 (243.3) and 1000 grains weight (183.8 g) were noted in control plots.

Growth parameters of maize significantly increased in treatments

containing Thiobacilli or S and the increase was more when Thiobacillus or S

were jointly used with or without P. Reason was the extent of bacterial S oxidation

mechanism that enhanced P, S and micronutrients bioavailability in the soil.

Significant relationship between soil P bioavailability and yield parameters of

maize was also identified by Khan et al. (2005) who concluded that the

application of high dose of P to maize crop plants increased number of cobs plant-

1, grain weight cob-1, number of grains cob-1 and 1000-grains weight. Similarly,

144

the findings made by Maqsood et al. (2001) indicated that high quantity of P

significantly effect number of cobs plant-1, number of grains plant-1 and 1000

grains weight in maize crop. Likewise, a positive significant increase in crop yield

parameters was also reported by Malhi and Gill (2002) as a consequent of high

dose of S application.

4.3.13 Treatment Effect on Yield Attributes of Maize

Data concerning grain, stalk and biological yields of spring and autumn

maize crops are given in Table 46. Increasing trend of grain, stalk and biological

yields in treatments was similar as was observed in case of growth parameters of

maize crop in the previous Table 45. Treatment P1S IW16 (Thiobacillus strain

IW16 was inoculated with 100 kg S ha-1 and 45 kg P2O5 ha-1) depicted the highest

values of grain (6.83 Mg ha1), stalk (14.06 Mg ha1) and biological yields (20.89

Mg ha1), while control treatment represented the lowest quantities of grain (1.71

Mg ha1), stalk (6.23 Mg ha1), and biological yields in spring maize crop. In

autumn maize crop treatment S IW16 (Thiobacillus strain IW16 plus 100 kg S

ha-1) contained maximum quantities of grain (7.24 Mg ha-1), stalk (14.25 Mg ha-1)

and biological yields (21.49 Mg ha-1), while control control plots achieved

minimum values of grain (1.59 Mg ha-1), stalk (6.15 Mg ha-1), and biological

yields (7.74 Mg ha-1).

Positive significant correlation existed between bio-available P in soil and

yield attributes of maize. Treatments where high concentration of P was

solubilized and was available for plant growth due to bacterial S oxidation

145

contained high values of yield attributes of maize crop. Results reported by Afzal

and Bano (2008) and Yazdani et al. (2009) were quite similar with these results

who also determined an increase in grain, stalk and biological yields in maize crop

under the effect of phosphorous solubilazation microorganisms which solubilized

and increased P concentration in the soil.

4.3.14 Phosphorous Uptake by Maize

Maximum concentration of P in spring maize grain was recorded (0.288

%) in treatment S IW16 (Thiobacillus strain IW16 with 100 kg S ha-1) and the

highest amount of P in spring maize stalk was determined (0.101 %) in treatment

P1S IW16 (Thiobacillus strain IW16 with 100 kg S ha-1 and 45 kg P2O5 ha-1).

Similarly, the highest quantity of P uptake by spring maize grain and stalk was

noted as 19.53 and 14.20 kg ha-1, respectively in treatment S IW16 (Table 47).

Minimum values of P concentration in spring maize grain and stalk was noted as

0.092 and 0.021 %, respectively (P uptake by spring maize grain and stalk 1.57

and 1.31 kg ha-1, respectively) in control.

The highest concentration of P in autumn maize grain and stalk (0.310 and

0.112 %, respectively) was recorded with P1S IW16 (Table 48). Treatment S IW16

was also found statistically at par with P1S IW16. Maximum quantity of P uptake

by autumn maize grain and stalk was determined as 22.30 and 15.79 kg ha-1 with S

IW16 and P1S IW16, respectively. The lowest P concentration in autumn maize

grain and stalk (0.087 and 0.019 %, respectively) and subsequently, the lowest P

uptake by autumn maize grain and stalk (1.38 and 1.17 kg ha-1, respectively) was

146

Table 47. Phosphorous uptake by spring maize

Treatments


Grain Stalk Grain Stalk Total

----------- % ---------- ---------------- kg ha-1 ----------------

Control 0.092 i 0.021 i 1.57 i 1.31 k 2.88 j

IW16 0.181 fg 0.058 fg 8.15 fg 5.82 hi 13.96 f

SW2 0.169 h 0.053 gh 7.37 h 5.26 ij 12.63 hi

P1 0.172 gh 0.052 h 7.10 h 5.19 j 12.29 i

P1 IW16 0.195 e 0.064 e 8.97 e 6.53 g 15.50 e

P1 SW2 0.181 fg 0.055 fgh 7.93 g 5.43 ij 13.36 fg

P2 0.184 f 0.077 c 8.04 g 7.65 ef 15.69 e

P2 IW16 0.208 d 0.078 c 9.86 d 8.12 e 17.98 d

P2 SW2 0.191 ef 0.071 d 8.58 ef 7.14 f 15.72 e

S 0.166 h 0.058 fg 7.35 h 5.80 hi 13.15 gh

S IW16 0.288 a 0.097 a 19.53 a 13.58 b 33.11 a

S SW2 0.252 c 0.085 b 16.05 c 11.64 c 27.69 b

P1S 0.171 gh 0.061 ef 7.63 gh 6.14 gh 13.77 fg

P1S IW16 0.274 b 0.101 a 18.71 b 14.20 a 32.91 a

P1S SW2 0.247 c 0.080 c 15.86 c 11.04 d 26.90 c

LSD 0.011 0.005 0.51 0.56 0.61


147

Table 48. Phosphorous uptake by autumn maize

Treatments


Grain Stalk Grain Stalk Total

---------- % ---------- ------------------ kg ha-1---------------

Control 0.087 i 0.019 j 1.38 j 1.17 i 2.55 j

IW16 0.198 e 0.068 g 9.19 e 7.08 f 16.27 f

SW2 0.180 g 0.059 h 7.92 gh 5.87 g 13.79 h

P1 0.168 h 0.051 i 7.27 i 5.06 h 12.33 i

P1 IW16 0.209 d 0.076 ef 9.74 d 7.93 e 17.67 e

P1 SW2 0.189 f 0.067 g 8.37 fg 6.69 f 15.06 g

P2 0.179 g 0.072 fg 7.64 h 7.07 f 14.71 g

P2 IW16 0.219 c 0.089 d 10.99 c 9.91 d 20.91 d

P2 SW2 0.197 e 0.080 e 9.08 e 8.28 e 17.36 e

S 0.177 g 0.071 fg 7.95 gh 7.13 f 15.08 g

S IW16 0.308 a 0.109 a 22.30 a 15.53 a 37.83 a

S SW2 0.255 b 0.097 c 17.24 b 13.42 c 30.66 c

P1S 0.188 f 0.069 g 8.48 f 6.95 f 15.43 fg

P1S IW16 0.310 a 0.112 a 21.89 a 15.79 a 37.68 a

P1S SW2 0.259 b 0.103 b 17.59 b 14.29 b 31.88 b

LSD 0.007 0.005 0.46 0.78 0.90


148

noted in control. plots The amount of P in autumn maize grain and stalk in control

plots was recorded less than the spring crop due to continuous P uptake by

consecutive two crops without P solubilization or addition. Similarly, positive

significant relationship between the quantity of plant available P in soil and P

concentration in maize grain and stalk was also reported by Delong et al. (2001)

and Alam and Shah (2002).

4.3.15 Correlation among Various Soil and Maize Variables

Quantities of bio-available P in the soil solution had a positive significant

correlation with all maize crop yield attributes both in spring and autumn crops

(Table 49 and Table 50). High concentrationt of bio-available P in the soil solution

increased total P uptake, growth parameters (cobs plant-1, grains plant-1, 1000

grain weight) and yield attributes (grain, stalk and biological yields, and harvest

index) of both the spring and autumn maize crops linearly. Figures 19, 20, 21 and

22 showed that bio-available P contents in soil, P uptake by maize crop and yield

attributes of maize crop are interlinked, interconnected and inter-dependable as

reported by Afzal and Bano, 2008 and Yazdani et al., 2009. Likewise, Alam et al.

(2003) and Rahman et al. (2007) recorded positive relationship between the

amount of total P uptake with all maize crop yield attributes like maize grain, stalk

and biological yields, and Harvest index.

149

y = 2.0877x + 1.8532

R2 = 0.92

0

8

16

24

32

40

0 4 8 12 16 20


Tot

al P

upt

ake

(kg

ha-1

)

y = 0.301x + 2.5336

R2 = 0.80

0

2

3

5

6

8

0 4 8 12 16 20


Mai

ze g

rain

yie

ld (

Mg

ha-1)

y = 0.5084x + 6.9261

R2 = 0.85

5

7

9

11

13

15

0 4 8 12 16 20


Mai

ze s

talk

yie

ld (

Mg

ha-1)

y = 0.8097x + 9.4567

R2 = 0.84

5

9

13

17

21

25

0 4 8 12 16 20


Bio

logi

cal y

ield

(M

g ha-1

)

Figure 19. Soil bio-available P relation with different spring maize yield attributes

150

y = 0.15x + 2.1626

R2 = 0.94

0

2

3

5

6

8

0 8 16 24 32 40


Mai

ze g

rain

yie

ld (

Mg

ha-1)

y = 0.2474x + 6.4053

R2 = 0.95

5

7

9

11

13

15

0 8 16 24 32 40


Mai

ze s

talk

yie

ld (

Mg

ha-1)

y = 0.3975x + 8.5649

R2 = 0.95

5

9

13

17

21

25

0 8 16 24 32 40


Bio

logi

cal y

ield

(M

g ha-1

)

y = 0.2213x + 26.522

R2 = 0.52

20

24

28

32

36

40

0 8 16 24 32 40


Har

vest

inde

x (%

)

Figure 20. Relation of P uptake with different spring maize yield attributes

151

y = 2.051x + 2.8213

R2 = 0.94

0

8

16

24

32

40

0 4 8 12 16 20


Tot

al P

upt

ake

(kg

ha-1

)

y = 0.28x + 2.6342

R2 = 0.82

0

2

3

5

6

8

0 4 8 12 16 20


Mai

ze g

rain

yie

ld (

Mg

ha-1)

y = 0.4236x + 7.4245

R2 = 0.84

5

7

9

11

13

15

0 4 8 12 16 20


Mai

ze s

talk

yie

ld (

Mg

ha-1)

y = 0.7037x + 10.059

R2 = 0.84

5

9

13

17

21

25

0 4 8 12 16 20


Bio

logi

cal y

ield

(M

g ha-1

)

Figure 21. Soil bio-available P relation with different autumn maize yield

attributes

152

y = 0.1423x + 2.1342

R2 = 0.95

0

2

3

5

6

8

0 8 16 24 32 40


Mai

ze g

rain

yie

ld (

Mg

ha-1)

y = 0.2132x + 6.7083

R2 = 0.96

5

7

9

11

13

15

0 8 16 24 32 40

Total P uptake (kg ha-1)M

aize

stal

kyi

eld

(Mg

ha-1)

y = 0.3555x + 8.8426

R2 = 0.96

5

9

13

17

21

25

0 8 16 24 32 40


Bio

logi

cal y

ield

(M

g ha-1

)

y = 0.2389x + 25.867

R2 = 0.61

20

24

28

32

36

40

0 8 16 24 32 40


Har

vest

inde

x(%

)

Figure 22. Phosphorous uptake relation with different autumn maize yield

attributes

153

Table 49. Correlation among various maize attributes

Spring maize

Parameters Bioavailable P

Total P uptake

Maize grain yield

Maize stalk yield

Biological yield


Maize grain yield

0.90** 0.97**

Maize stalk yield

0.92** 0.98** 0.99**

Biological yield

0.91** 0.98** 0.99** 0.99**

Harvest index 0.54** 0.72** 0.84** 0.78** 0.80**

Autumn maize


Maize grain yield

0.91** 0.98** 0.95**

Maize stalk yield

0.92** 0.98** 0.95** 0.99**

Biological yield

0.91** 0.98** 0.95** 0.99**

Harvest index 0.63** 0.78** 0.72** 0.89**

* p ≥ F 0.05 ** p ≥ F 0.01

154

Table 50. Linear regression analyses among soil and maize variables


(R2)

Spring maize

TPU vs. BAP y = 2.0877 BAP + 1.8532 0.92

1000 GW vs. BAP y = 7.6354 BAP + 200.73 0.81

MGY vs. BAP y = 0.301 BAP + 2.5336 0.80

MSY vs. BAP y = 0.5084 BAP + 6.9261 0.85

BY vs. BAP y = 0.8097 BAP + 9.4567 0.84

MGY vs. TPU y = 0.15 TPU + 2.1626 0.94

MSY vs. TPU y = 0.2474 TPU + 6.4053 0.95

BY vs. TPU y = 0.3975 TPU + 8.5649 0.95

Autumn maize

TPU vs. BAP y = 2.0546 BAP - 3.3817 0.96

1000 GW vs. BAP y = 0.611 BAP + 39.881 0.95

MGY vs. BAP y = 0.2365 BAP + 1.4353 0.84

MSY vs. BAP y = 0.2009 BAP + 1.8812 0.78

BY vs. BAP y = 0.4373 BAP + 3.3165 0.81

MGY vs. TPU y = 0.1423 TPU + 2.1342 0.95

MSY vs. TPU y = 0.2132 TPU + 6.7083 0.96

BY vs. TPU y = 0.3555 TPU + 8.8426 0.96

BAP: Bioavailable P, TPU: Total P uptake, MGY: Maize grain yield, MSY: Maize stalk yield, BY: Biological yield, HI: Harvest index

155

SUMMERY

The study evaluated the capacity of Thiobacillus spp. with or without S

and P fertilizer for enhancing P bioavailability in soil through S oxidation.

Samples were collected from different ecologies; SOB isolates were isolated and

screened on the basis pH reduction in thiosulphate broth media, colour change in

thiosulphate agar plates, phosphorous solubilization index and their P-

solubilization efficiency through bioleaching tests in broth containing tricalcium

phosphate and phosphate rock. Seven most efiicient isolates were identified as

genus Thiobacillus. Four most efficient Thiobacillus isolates were inoculated in

alkaline and calcareous soil in combination with three S levels S1, S2 and S3 (25,

37.5 and 50 mg kg-1, respectively) to determine pH, ECe, CaCO3 contents,

sequential P fractions, bio-available P, different S fractions and micronutrients in

the incubated soil after 30, 60 and 90 days of incubation. Two field experiments

were conducted under two permanent lay outs and the two best Thiobacillus

strains with S 100 kg ha-1 were inoculated in combination with two doses of P

fertilizer viz., 45 and 90 kg P2O5 ha-1. Two cropping systems (rice-wheat and

maize-maize) were tested to determine P uptake and the amount of bio-available P

in soil. Growth and yield attributes of crops were also recorded.

The findings of the present study are as under:

The most efficient SOB isolates were found in sulfur based ecologies such as

industrial wastewater, sulfur mud and sewerage water.

155

156

The genus Thiobacillus of SOB was found efficient in sulfates production

through S oxidation and dropped pH significantly in the growth media.

The quantity of P dissolved in thiosulphate tricalcium phosphate and rock

phosphate media had a significant positive correlation with the concentration of

biologically produced sulfates by Thiobacillus spp.

During soil incubation study maximum solubilization of two insoluble

calcium bounded P compounds Ca8-P and Ca10-P (net decrease of 10.18 and 4.72

mg kg-1, respectively) was recorded where Thiobacillus isolate IW16 was

inoculated with 50 mg S kg-1, whereas P solubilization phenomenon increased the

concentration of sparingly soluble Ca2-P (net increase of 20.33 mg kg-1) and bio-

available P contents (net increase of 22.26 mg kg-1) in the soil simultaneously.

Bioavailability of soil micronutrients increased linearly as a result of reduction in

pH and CaCO3 contents in the soil.

During rice and wheat crops the concentration of bio-available P increased

(net increase of 11.90 mg kg-1) with Thiobacillus isolate IW16 in combination

with 100 kg S ha-1. Phosphorous uptake by rice and wheat plants was positively

and significantly affected by soil bio-available P contents.

Combined effect of Thiobacillus strain IW16 and S attained the maximum

net increased amount of bio-available P in the soil solution after spring and

autumn maize harvest (10.34 and 12.35 mg kg-1, respectively) and had positive

significant correlation with the concentration of P in maize plants.

Yield and growth attributes of rice, wheat and maize crops had high positive

significant correlation with P solubilzation and enhancement in soil.

157

CONCLUSIONS

a. Best ecologies of SOB are sulfur based such as industrial wastewater,

sulfur mud and sewerage water.

b. The Genus Thiobacillus of SOB is extremely competent in sulfuric acid

production and proved its worth in pH reduction in different media such

as thiosulphate, thiosulphate tricalcium phosphate, rock phosphate and

soil by efficiently oxidizing S into sulfuric acid.

c. Bacterial sulfur oxidation mechanism is a sulfuric acid generating

phenomenon. This bacterially produced sulfuric acid has a key role in P

solubilization from different fixed forms of P present in rock phosphate

or in soil. Insoluble calcium bounded P compounds (Ca8-P and Ca10-P)

were significantly dissolved and converted into bio-available P forms by

SOB through S oxidation.

d. Amount of P dissolution and enhancement has a massive positive

significant correlation with the concentration of biologically produced

sulfuric acid.

e. Interaction between Thiobacillus spp. and S was highly significant in

enhancing bio-available P contents in soil and boosting all growth and

yield parameters of crops.

f. Soil treatment with Thiobacilli in combination with S was the best

approach to improve soil P fertility by solubilizing already present huge

quantity of fixed P in the soil.

158

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Appendices Appendix I. Description of samples for the isolation of SOB 1. Paddy fields (PF)

Sr. # Microbial Ecology Identification

name

1. Sunder area (Lahore) PF1




5. Sahianwala area PF5





10. Sheikhupur area PF10






2. Wheat Rhizosphere


name

1. Sahianwala area WR1





201




9. Herdaive (Shekhupura) WR9
















25. Chung Area (Lahore) WR25

26. Mohenalwal area (Lahore) WR26







33. PMAS Arid Agriculture University Rawalpindi WR33


202


36. Gujranawala area WR36










46. Gagher area WR46





2. Sugarcane Rhizosphere (SR)


name

1. Herdaive (Shekhupura) SR1








9. Gujranwala Khiali bypass area SR9


203



13. Mohenalwal area (Lahore) SR13



4. Maize Rhizosphere


name

1. Sahianwala area MR1




5. Chung Area (Lahore) MR5




9. Gujranwala Khiali bypass area MR9



12. Mohenalwal area (Lahore) MR12




5. Industrial wastewater


name

1. Sunder area (Lahore) IW1



204



6. Faisalabad area IW6







13. Sulfuric Acid producing Factory area (Lahore) IW13



6. Canal water (CW)


name

1. Sukheyky area CW1



4. Duska CW4

5. Duska CW5

6. Duska CW6

7. Duska CW7

8. Gujrat CW8

9. Gujrat CW9

10. Gujrat CW10

7. Sulfur mud


name

1. Sulfuric Acid producing Factory area (Lahore) SM1

205















8. Sewage water


name

1. Sukheyky area SW1





6. Faisalabad area SW6





11. Chung Area (Lahore) SW11


206



15. Gujranwala Khiali bypass area SW15

9. Industrial waste sludge


name

1. Sunder area (Lahore) IS1






7. Thoker area (Lahore) IS7









10. Sewage sludge


name

1. Sukheyky area SS1





207


7. Gujranwala Khiali bypass area SS7




Appendix II. pH reduction by sulfur oxidizing bacterial isolates

Treatments Incubation days

Net reduction 01 02 04 08 16

Control 8.00 a 8.00 a 8.00 a 8.00 a 8.00 a 0.00

PF2 7.81 d 7.24 no 6.93 m 6.54 m 6.13 p 1.87

PF3 7.93 bc 7.62 gh 7.34 h 7.12 d 6.91 de 1.09

IW1 7.14 k 6.63 v 5.42 x 3.13 z 2.84 ] 5.16

IW3 7.75 e 7.11 qrs 6.73 o 6.25 p 5.91 qr 2.09

IW4 7.94 bc 7.45 kl 7.14 k 6.67 ijk 6.35 j 1.65

IW5 7.93 bc 7.32 mn 6.92 m 6.63 kl 6.24 mn 1.76

IS1 7.84 d 7.21 op 6.71 o 6.35 o 5.91 qr 2.09

IS2 7.71 e 7.05 rs 6.54 r 6.17 pqr 5.86 qrs 2.14

IS11 7.83 d 7.34 m 6.82 n 6.54 m 6.27 klm 1.73

IS12 7.91 bc 7.53 jk 7.23 ij 6.87 fg 6.43 i 1.57

IS16 7.93 bc 7.74 cd 7.44 ef 7.01 e 6.85 ef 1.15

SW1 7.84 d 7.35 m 6.65 op 6.43 n 5.92 qr 2.08

SW2 7.04 l 6.55 w 5.23 y 2.84 [ 2.63 ^ 5.37

208

SW4 7.63 f 7.04 s 6.45 st 6.12 rs 5.85 rs 2.15

SS1 7.42 h 7.05 rs 5.93 u 4.73 v 4.46 y 3.54

SW5 7.84 d 7.23 o 6.91 m 6.67 ijk 6.32 jkl 1.68

CW1 7.92 bc 7.63 gh 7.45 ef 7.24 c 6.81 f 1.19

CW2 7.91 bc 7.25 no 6.93 m 6.73 hi 6.23 mno 1.77

CW3 7.83 d 7.32 mn 6.62 pq 6.24 pq 5.94 q 2.06

SS4 7.95 abc 7.65 efg 7.55 cd 7.25 c 6.93 d 1.07

SS6 7.93 bc 7.44 l 7.23 ij 6.73 hi 6.45 hi 1.55

WR2 7.94 bc 7.46 kl 7.06 l 6.64 jk 6.16 op 1.84

WR4 7.65 f 7.13 qr 6.57 qr 6.16 qr 5.52 v 2.48

WR7 7.92 bc 7.52 jk 7.18 ijk 6.71 hij 6.15 p 1.85

IW7 7.91 bc 7.73 cde 7.52 de 7.06 de 6.73 g 1.27

WR9 7.94 bc 7.54 ij 7.19 ijk 6.77 h 6.27 j-m 1.73

WR10 7.65 f 7.14 pq 6.52 rs 6.23 pq 5.82 s 2.18

WR12 7.43 h 7.06 qrs 6.43 t 6.04 t 5.63 u 2.37

WR13 7.74 e 7.26 no 6.53 r 6.35 o 5.94 q 2.06

WR14 7.92 bc 7.63 gh 7.44 ef 6.51 m 6.30 j-m 1.70

WR15 7.91 bc 7.54 ij 7.16 jk 6.66 ijk 6.52 h 1.48

WR16 7.94 bc 7.79 bc 7.61 bc 7.23 c 7.01 bc 0.99

SM2 7.92 bc 7.73 cde 7.52 de 7.08 de 6.84 ef 1.16

SM3 7.54 g 6.92 t 6.43 t 6.06 st 5.31 w 2.69

SR2 7.91 bc 7.54 ij 7.36 gh 6.93 f 6.30 j-m 1.70

209

SR8 7.92 bc 7.55 hij 7.14 k 6.72 hij 6.25 lm 1.75

IW13 7.33 i 6.83 u 5.73 w 3.87 x 3.53 [ 4.47

IW14 7.24 j 6.82 u 5.72 w 3.44 y 3.08 \ 4.92

IW16 6.93 m 6.23 x 5.08 z 2.65 \ 2.42 _ 5.58

SM1 7.32 i 6.92 t 5.81 v 4.13 w 3.74 z 4.26

SM4 7.90 c 7.71 c-f 7.53 d 7.25 c 6.95 cd 1.05

SW11 7.54 g 6.83 u 6.44 t 5.63 u 5.12 x 2.88

SW14 7.96 abc 7.61 ghi 7.25 i 6.65 ijk 6.35 jk 1.65

MR6 7.94 bc 7.52 jkl 7.33 h 6.72 hij 6.46 hi 1.54

MR8 7.93 bc 7.45 kl 7.06 l 6.56 lm 6.17 nop 1.83

SM11 7.51 g 7.13 qr 6.54 r 6.24 pq 5.73 t 2.27

SM7 7.91 bc 7.68 d-g 7.42 fg 7.13 d 6.95 cd 1.05

SM9 7.97 ab 7.84 b 7.65 b 7.36 b 7.06 b 0.94

SM12 7.95 abc 7.73 cde 7.52 de 7.06 de 6.82 f 1.18

SM14 7.93 bc 7.64 fg 7.33 h 6.84 g 6.47 hi 1.53

LSD 0.051 0.072 0.072 0.072 0.072

p ≥ F 0.05. Similar letter (s) values in a column are not statistically different Appendix III. Colour change by SOB isolates


01 02 04 08

------------------------------ (%) ---------------------------------

Control 0.00 h 0.00 t 0.00 w 0.00 s

210

PF2 0.00 h 11.30 l 23.65 p 62.45 l

IW1 33.35 c 81.55 b 100.00 a -

IW3 0.00 h 9.40 s 41.50 l 82.75 f

IW5 0.00 h 0.00 t 11.70 s 53.65 o

IS1 0.00 h 12.25 i 39.50 o 81.40 h

IS2 0.00 h 11.55 j 42.65 i 79.35 j

IS11 0.00 h 0.00 t 11.20 u 50.35 q

SW1 0.00 h 9.75 q 41.85 j 81.50 g

SW2 41.30 b 100.00 a - -

SW4 0.00 h 12.85 h 21.60 r 63.35 k

SS1 9.75 g 41.30 e 81.25 c 100.00 a

SW5 0.00 h 0.00 t 11.45 t 43.65 r

CW2 0.00 h 0.00 t 9.20 v 54.45 n

CW3 0.00 h 11.45 k 44.80 f 80.35 i

WR2 0.00 h 9.50 r 22.65 q 61.45 m

WR4 0.00 h 10.85 m 43.15 h -

WR10 0.00 h 10.35 p 41.65 k 92.15 b

WR12 0.00 h 10.45 o 43.70 g 100.00 a

WR13 0.00 h 10.65 n 40.95 m 90.85 e

SM3 0.00 h 23.10 f 49.40 e 100.00 a

IW13 10.15 f 41.25 e 82.65 b 100.00 a

IW14 21.85 d 63.35 c 100.00 a -

211

IW16 43.70 a 100.00 a - -

SM1 21.35 e 62.15 d 100.00 a -

SW11 0.00 h 22.85 g 49.55 d 100.00 a

MR8 0.00 h 0.00 t 11.15 u 52.10 p

SM11 0.00 h 11.35 l 39.75 n 91.20 d

LSD 0.052 0.073 0.090 0.073


Appendix IV. Phosphorous solubilization index of SOB isolates


01 02 04 08

Control 0.00 h 0.00 t 0.00 y 0.00 w

PF2 0.00 h 0.00 t 1.61 s 1.64 st

IW1 1.71 c 3.24 c 5.25 e 7.12 c

IW3 0.00 h 0.47 r 1.80 pq 1.80 q

IW5 0.00 h 0.00 t 1.00 w 0.88 v

IS1 0.00 h 0.67 q 1.86 p 1.93 r

IS2 0.00 h 0.41 s 1.75 qr 1.76 qr

IS11 0.00 h 0.00 t 1.15 v 1.28 u

SW1 0.00 h 0.73 p 2.10 o 2.14 o

SW2 2.50 b 4.23 b 7.15 b 8.42 b

SW4 0.00 h 0.00 t 1.70 r 1.70 rs

212

SS1 0.53 g 2.10 g 4.42 f 4.72 g

SW5 0.00 h 0.00 t 0.81 x 0.86 v

CW2 0.00 h 0.00 t 1.25 u 1.32 u

CW3 0.00 h 0.85 o 2.26 n 2.34 n

WR2 0.00 h 0.00 t 1.71 r 1.74 qr

WR4 0.00 h 1.12 l 2.83 k 2.72 l

WR10 0.00 h 1.21 k 3.00 j 3.24 k

WR12 0.00 h 1.37 j 3.1 i 3.35 j

WR13 0.00 h 0.91 n 2.53 m 2.58 m

SM3 0.00 h 1.62 i 3.30 h 3.76 i

IW13 0.67 f 2.32 f 5.22 e 5.56 f

IW14 1.22 d 2.42 e 6.53 c 6.86 d

IW16 3.52 a 5.33 a 8.25 a 9.83 a

SM1 0.95 e 2.67 d 5.64 d 5.93 e

SW11 0.00 h 1.87 h 3.63 g 4.10 h

MR8 0.00 h 0.00 t 1.43 t 1.58 t

SM11 0.00 h 1.00 m 2.71 l 2.74 l

LSD 0.052 0.052 0.073 0.090


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