Avinash M.SC Thesis

A STUDY ON CHLORPYRIFOS DEGRADING BACTERIA
UNDER RICE (Oryza sativa L.) ECOSYSTEM
AVINASH, S. H.
DEPARTMENT OF AGRICULTURAL MICROBIOLOGY

COLLEGE OF AGRICULTURE, RAICHUR
UNIVERSITY OF AGRICULTURAL SCIENCES
RAICHUR – 584 104
JULY, 2018
A STUDY ON CHLORPYRIFOS DEGRADING BACTERIA
UNDER RICE (Oryza sativa L.) ECOSYSTEM
Thesis submitted to the

University of Agricultural Sciences, Raichur
In partial Fulfilment of the requirements for the
Degree of
Master of Science (Agriculture)
IN
AGRICULTURAL MICROBIOLOGY
By
AVINASH, S. H.

RAICHUR – 584 104
JULY, 2018
RAICHUR - 584 104
CERTIFICATE
This is to certify that the thesis entitled “A STUDY ON CHLORPYRIFOS

DEGRADING BACTERIA UNDER RICE (Oryza sativa L.) ECOSYSTEM”
submitted by Mr. AVINASH, S. H. in partial fulfillment of the requirements for the
award of the degree of MASTER OF SCIENCE (Agriculture) in AGRICULTURAL
MICROBIOLOGY to the University of Agricultural Sciences, Raichur, is a record of
bonafide research work carried out by him during the period of his study in this university
under my guidance and supervision, and that no part of the thesis has been submitted for
the award of any other degree, diploma, associate ship, fellowship or other similar titles.
Place: RAICHUR __________________________

Date: JULY, 2018 (NAGARAJ M. NAIK)
MAJOR ADVISOR
Approved by
Chairman: __________________________
(NAGARAJ M. NAIK)
Members: 1. __________________________
(MAHADEVA SWAMY)
2. __________________________
(R. C. GUNDAPPAGOL)
3. __________________________
(M. BHEEMANNA)
4. __________________________
(HARISCHANDRA R. NAIK)
ACKNOWLEDGEMENT
“Education plays important role in personal and social development and teacher
plays a fundamental role in imparting education. Teachers have crucial role in preparing
young people not only to face the future with confidence but also to build up it with purpose
and responsibility. There is no substitute for teacher pupil relationship. I start in the name
of God-who has bestowed upon me all the physical and mental attributes that I possess and
skills to cut through and heal a fellow human”.
With immense pleasure and a sense of high reverence I express my sincere and
heartfelt gratitude to Dr. Nagaraj M. Naik, Assistant Professor, Department of
Agricultural Microbiology and Chairman of my Advisory Committee, for his inspiring
guidance, constructive criticism and timely advisement during the entire course of my
research. I am extremely thankful to him for his supervision, Knowledge and enthusiastic
interest, which he provided me throughout my post-graduation and research work.
I have immense pleasure in expressing my whole-hearted sense of gratitude and

appreciation for Dr. Mahadeva Swamy, Professor and Head (Agricultural Microbiology),
and member of my advisory committee for his blessings, inspiring suggestions, enthusiastic
interest and encouragement despite of his busy schedule of work which provided me solace
during the tenure of investigation.
I take this opportunity to express sincere heartily thanks to my Advisory Committee

members, Dr. R. C. Gundappagol, Senior Scientist, ARS, Kalaburgi, Dr. M. Bheemanna,
Professor and Head, PRFQAL (Department of Agricultural Entomology), UAS, Raichur
and Dr. Harischandra R. Naik, Assistant Professor, PRFQAL (Department of Agricultural
Entomology), UAS, Raichur. Grace of almighty has always been felt in having given me
these unique personalities to guide and inspire me with their sincere technically valuable
suggestions, untiring patience, concern, appreciations and constant encouragement.
A very sincere and warm hearted thanks to Mr. Balakrishna Gowda, Assistant
Professor, Department of Agricultural Microbiology, AC, Raichur for more of his friendly
nature that encouraged me during my course work. I am also thankful to Dr. Tamil
Vendon, K., Professor, UAS, Bangalore, Dr. Narayana Rao, Professor and Head of the
Department of Soil Science, College of Agriculture, Raichur and our Department teaching
staff Mr. Pampana Gowda, Assistant Professor, Mrs. Shwetha Meti, Assistant Professor,
AC, Raichur, Dr. Yallappa, Assistant Professor, AC, Beemarayanagudi for their valuable
suggestions during the course of study.
I also express my profound sense of gratitude to Dr. I. Shankargoud, Professor and
Dean (PGS), UAS, Raichur and Dr. M. G. Patil, Professor and former Dean (PGS), UAS,
Raichur for constant support and Dr. B. S. Golasangi, Technical officer, UAS, Raichur for
improvising the thesis presentation.
I have been fortunate in having a person in whose company, I never felt the burden
of studies and her inspiring nature always held me from falling down & made me to grow
as a better person. The flower scented my life with elegant fragrance, Manasa.
I avail this opportunity to express my sincere gratitude to my dearest and beloved

friends Nagaraj, K., Nagraj, B., Arti, Asha, Savitri, Vijayalakshmi, Bindu and my senior
friends Swapna, Krishnaveni, Gagana, Suma, Sushma, Raghavendra as well as junior
friends Malappa, Shivleela, Sushma, Eramma, Sowmya, Sushma, and Mamtha for their
care and love.
Words can never express the gratitude to my beloved senior Y. Nagaraju, for
inspiring me in every aspect. A very Special, warm and heartfelt thanks Srikanth, Raju,
Raghu, Revu, Balu, Sumit, Farooq, Vinayak, Praveen and Kiran who never been less than
my brothers and made me smile all along and always been there with me.
And also, I specially thank Rahul, Mr. Pavan, SRF, PRFQAL, Mr. Chandrashekhar,
SRF, PRFQAL, and Sri. Anjanayya, Lab Assistant, Department of Agricultural
Microbiology, AC, Raichur, for their help, support and encouragement.
Words can hardly express the heartfelt gratitude to my beloved parents whose
selfless love, affection, obstinate sacrifices and blessing made my path easier. I would like
to convey my cordial thanks to all those unmentioned personalities who helped me directly
and indirectly to fulfill my dream. Finally, I am greatly thankful to the Lord for giving me
this beautiful life and always illuminating me with the light of success.
Place: Raichur
JULY, 2018 (AVINASH, S. H.)
CONTENTS
Sl. Page
Chapter Particulars
No. No.
CERTIFICATE iii
ACKNOWLEDGEMENT iv
LIST OF ABBREVIATIONS v
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF PLATES viii
LIST OF APPENDICES ix
I INTRODUCTION 1-4
II REVIEW OF LITERATURE 5-23
Isolation and screening the efficient chlorpyrifos degrading

2.1 5
bacteria
Characterization of the efficient chlorpyrifos degrading

2.2 11
bacterial isolates
In vitro screening of the bacterial isolates for their beneficial

2.3 12
attributes
Biodegradation analysis of the chlorpyrifos degrading

2.4 19
bacterial strains
Evaluation of the effect of efficient pesticide degrading

2.5 22
bacterial isolates under in vivo conditions
III MATERIAL AND METHODS 24-40
Collection of soil samples for the isolation of chlorpyrifos

3.1 24
degrading bacteria
3.2 Characterization of soil samples for chemical properties 24
Isolation and purification of chlorpyrifos degrading bacteria

3.3 26
from the rhizosphere of rice
In vitro screening for the growth of chlorpyrifos degrading

3.4 26
bacteria in MSM broth by enrichment culture technique
Estimation of per cent chlorpyrifos degradation by efficient

3.5 26
isolates under in vitro conditions
Sl. Page
Chapter Particulars
No. No.
3.6 Characterization of the efficient chlorpyrifos degrading 27

bacterial isolates.
3.7 In vitro screening of the efficient chlorpyrifos degrading 31

bacterial isolates for their beneficial attributes
3.8 Evaluation of efficient chlorpyrifos degrading bacterial 32

isolates on growth of paddy under pot culture
3.8.1 Preparation of inoculants 34
3.4.2 Planting pattern 34
3.4.3 Plant growth parameters 34
3.4.4 Determination of microbial population in the soil 35
3.4.5 Determination of enzymatic activity in the soil 36
Analysis of chlorpyrifos degradation in soil by the efficient

3.4.6 bacterial isolates using Gas Chromatography-Mass 37
Spectroscopy (GC-MS/MS)
IV EXPERIMENTAL RESULTS 41-87

4.1 41
degrading bacteria
4.2 Characterization of soil samples for chemical properties 41
Isolation and purification of chlorpyrifos degrading bacteria

4.3 41
from the rhizosphere of rice
In vitro screening for growth of chlorpyrifos degrading

4.4 bacterial isolates in minimal salt medium by enrichment 50
culture method
Estimation of percent chlorpyrifos degradation by efficient

4.5 50
isolates under in vitro conditions
Characterization of efficient chlorpyrifos degrading bacterial

4.6 53
isolates
In vitro screening of the efficient chlorpyrifos degrading

4.7 60
bacteria for their plant growth promoting attributes
Evaluation of efficient chlorpyrifos degrading bacteria on

4.8 66
growth parameters of rice under pot culture
Contd….
Sl. Page
Chapter Particulars
No. No.
4.8.1 Plant growth parameters 66
4.8.2 Soil enzymatic analysis 72
4.8.3 Determination of microbial population in the soil 75
Analysis of chlorpyrifos degradation in soil by the efficient

4.8.4 80
bacterial isolates using GC-MS/MS
V DISCUSSION 88-117

5.1 89
degrading bacteria
5.2 Physico-chemical analysis of the soil samples 89
Isolation and purification of the chlorpyrifos degrading

5.3 90
bacteria from the rhizospheric soils of rice
In vitro screening for growth of chlorpyrifos degrading

5.4 bacterial isolates in minimal salt medium by enrichment 91
culture method
Estimation of percent chlorpyrifos degradation by the

5.5 91
efficient isolates under in vitro conditions
Characterization of the efficient chlorpyrifos degrading

5.6 94
bacterial isolates
In vitro screening of efficient chlorpyrifos degrading bacteria

5.7 97
for their plant growth promoting attributes
Evaluation of efficient chlorpyrifos degrading bacteria on the

5.8 101
growth of paddy under pot culture
VI SUMMARY AND CONCLUSIONS 118-121
VII REFERENCES 122-135
APPENDIX 136-139
LIST OF ABBRIVATIONS
Abbreviation Expansion
% Per cent
& And
/ Otherwise or per
i.e. that is
ANOVA Analysis of variance
BLQ Below quantification level
CRD Completely randomized design
CFU Colony forming units
C.D. Critical Difference
cm Centimeter/s
Conc. Concentration
C.V. Co efficient variation
DAT Days after Transplanting
DF Degrees of freedom
DW Dry weight
dSm-1 Deci Simon per meter
EC Electrical conductivity
et. al., Group of workers
FYM Farm yard manure
Fig. Figure
g Gram/s
ha Hectare/s
GC-MS/MS Gas Chromatography Mass Spectroscopy
hr. Hour
J. Journal
kg Kilogram/s
kg ha-1 Kilograms per hectare
LOQ Limit of Quantification
MSM Minimal Salt Media
µg Microgram
m ha million hectares
mt million tonnes
mg Milligram
mm millimeter
No Number
°
C Centigrade
OC Organic carbon
P Phosphorus
Ppm Parts per million
ppb Parts per billion
Res. Research
S. Em. Standard Error of mean
Sci. Science
t ha-1 Tonnes per hactare
TPF 1.3.5-triphenylformazan
Viz., Namely
Wt. Weight
LIST OF TABLEs
Table Page
Title
No. No.
1 Treatment details of the pot culture experiment 33

Details of location, soil type and source of soil samples used for
2 42
isolation of chlorpyrifos degrading bacteria
Chemical properties of paddy rhizospheric soil samples collected
3 44
from Tungabhadra command area
In vitro screening for growth of chlorpyrifos degrading bacterial
4 isolates in minimal salt medium supplemented with 300 ppm CP 46
concentration
Invitro screening for growth of chlorpyrifos degrading bacterial
concentration
Invitro screening for growth of chlorpyrifos degrading bacterial
concentration
Degradation percentage of chlorpyrifos at 200 ppm concentration
7 in MSM broth inoculated with efficient bacterial isolates at 54
different time intervals
Morphological characteristics of the efficient chlorpyrifos
8 55
degrading bacterial isolates isolated from rhizosphere soil of rice
Biochemical characteristics of the efficient chlorpyrifos degrading
9 57
bacterial isolates isolated from rhizosphere soil of rice
IAA and siderophore production by the efficient chlorpyrifos
10 62
degrading bacterial isolates
Phosphate solubilization by efficient chlorpyrifos degrading
11 64
bacterial isolates
Plant height of rice as influenced by the application of efficient
12 67
chlorpyrifos degrading bacterial inoculants
Number of tillers per hill of rice as influenced by the application of
13 69
efficient chlorpyrifos degrading bacterial inoculants
Number of leaves per hill of rice as influenced by the application of
14 71
Total dry weight per hill of rice as influenced by the application of
15 73
Contd…..
Table Page
Title
No. No.
Dehydrogenase activity of the soil as influenced by the application

16 74
of efficient chlorpyrifos degrading bacterial inoculants
Phosphatase activity of the soil as influenced by the application of

17 76
Bacterial population of the soil as influenced by the application of

18 78
Fungal population of the soil as influenced by the application of

19 79
Population of actinomycetes in the soil as influenced by the

20 81
application of efficient chlorpyrifos degrading bacterial inoculants
Concentration and percent degradation of chlorpyrifos by the

21 83
efficient bacterial isolates estimated using GC-MS/MS
LIST OF FIGURES
Fig Page
Title
No. No.
1 Structure of chlorpyrifos 47
Google map showing the areas of soil samples collected for the 47
2
isolation of chlorpyrifos degrading bacteria
Linearity calibration curve for 0.01 to 0.1 mg kg-1 of chlorpyrifos 85

3a
in soil
Chromatogram of standard for 0.01 to 0.1 mg kg-1 of chlorpyrifos 85

3b
in soil
GCMS/MS Spectrum of chlorpyrifos from the soil samples at 50- 86

4
dilution
Residual concentration of chlorpyrifos at 200 ppm concentration in 92

5a
MSM broth inoculated with efficient bacterial isolates
Degradation percentage of chlorpyrifos at 200 ppm concentration 93

5b
in MSM broth inoculated with efficient bacterial isolates
IAA production by efficient chlorpyrifos degrading bacterial 99

6
isolates
Siderophore production by efficient chlorpyrifos degrading 100

7
bacterial isolates
Phosphate solubilization by efficient chlorpyrifos degrading 102

8
bacterial isolates
Plant height of rice as influenced by the application of efficient 104

9
Number of tillers per hill of rice as influenced by the application of 105

10
Number of leaves per hill of rice as influenced by the application of 106

11
Total dry weight per hill of rice as influenced by the application of 107
12
Dehydrogenase activity of the soil as influenced by the application 109

13
of efficient chlorpyrifos degrading bacterial inoculants
Phosphatase activity of the soil as influenced by the application of 110

14
Contd…..
Fig Page
Title
No. No.
Bacterial population of the soil as influenced by the application of

15 112
Fungal population of the soil as influenced by the application of

16 113
Population of actinomycetes in the soil as influenced by the

17 114
application of efficient chlorpyrifos degrading bacterial inoculants
Chlorpyrifos degradation analysis using GC-MS/MS after 30 days

18 116
of application
LIST OF PLATES
Plate Page
Title
No. No.
1 Isolated colonies of chlorpyrifos degrading bacteria on MSM agar 48
2 Purification of the chlorpyrifos degrading bacterial isolates 48
3 Pure culture slants of the isolated 25 chlorpyrifos degrading bacteria 49
Hallow zones around the colonies showing positive for starch

4 58
hydrolysis test
5 Production of foam indicating catalase activity 58
Blue coloration of the media showing positive for citrate utilization

6 59
test
7 Yellow colouration in MR-VP broth indicating negative for VP test 59
8 Production of IAA by the efficient chlorpyrifos degrading bacteria 63
Production of siderophore by the efficient chlorpyrifos degrading

9 63
bacteria
Phosphate solubilization by the efficient chlorpyrifos degrading

10 65
bacteria
11 Overall view of pot culture experiment 65
12 GC-MS/MS instrument used in chlorpyrifos degradation analysis 84

LIST OF APPENDICES
Appendix Page
Title
No. No.
I Composition of the media 136

Introduction
I. INTRODUCTION
Rice (Oryza sativa L.) is one of the important food grains and the largest crop grown
in the world in terms of both area and production. More than half the world population
depends on rice, especially in developing countries. It provides about 22 % of calories and
17 % of protein. Globally, rice is cultivated in an area about 161.10 million hectare and
production of about 487.50 million tons with the productivity of 3.14 tons per hectare
(www.statista.com). In India, rice is being cultivated with an area of 43.993 million hectares
and ranks second in production (109.698 million tons) next to China. The average yield of
rice in India is 2494 kg/ha. In Karnataka, rice is being cultivated with an area of 1.03 million
hectare with the production 2.604 million tons. The average yield of rice in Karnataka is
2519 kg/ha (www.indianstat.com).
The pesticides are the important part of crop protection in the cultivation of rice.
They belong to a category of chemicals used worldwide to prevent or control pests,
diseases, weeds and other plant pathogens in an effort to reduce or eliminate yield losses
and to maintain high product quality. The positive aspect of the application of pesticides
has resulted in enhanced crop/food productivity and a drastic reduction of vector-borne
diseases.
The pesticides were introduced in agriculture to fulfil the increased food needs
of growing population. But now, the use of pesticides has become a necessary evil.
Residues of applied pesticides stay in the environment (air, soil, ground and surface water)
for variable periods of time. Due to the long persistence, tendency to bio-accumulate and
potential toxicity towards non-target organisms, organochlorines group of pesticides was
replaced by relatively less persistent and yet effective organophosphorus (OP) compounds.
The organophosphorus pesticides are biodegradable organic compounds containing

carbon-phosphorus bonds; used primarily in pest control. Environmental pollution caused
by pesticides and their degradation products is a major ecological problem (Galloway and
Handy, 2003). It has been documented that organophosphorus pesticides (OP) constitute
the largest group of pesticides used globally account for about 38 % of the total pesticides
used worldwide (Singh et al., 2006).
The extensive application of organophosphorus (OP) insecticides such as
chlorpyrifos, phorate, malathion and dichlorvos are employed for plant protection against
insect pests. These pesticides are one of the major chemicals responsible for the
contamination and deterioration of soil and groundwater, particularly in the close vicinities
of agricultural fields. Considering their high toxicity and persistence in the environment,
most of them are banned all over the world.
Organophosphorus insecticides are being increasingly used in rice as a substitute

for organochlorine and carbamate insecticides because of their high efficiency.
Chlorpyrifos [O,O-diethyl-O-(3-5-6-trichloro-2-pyridyl) phosphorothioate], a
phosphonothioate insecticide, has been commercially used since the 1960s. Globally,
chlorpyrifos ranks first among the conventional pesticide active ingredients in the
agricultural sector with the production of 3.64 to 4.99 million kg during 2007 (Grube et al.,
2011). In India, chlorpyrifos was the second most used agricultural insecticide in 2013-14
at 9540 tons of formulation (Ministry of Chemicals and Fertilizers, Govt. of India, 2014).
Chlorpyrifos (CP) is used particularly for the control of broad-spectrum of insect

pests in economically important crops. Chlorpyrifos is intensively used for effective control
of gall midge (Orseolia oryzae), leaf-folder (Cnaphalocrocis medinalis) and leafhopper
(Nephotettix virescens) in rice, and soil applications to control termites (Reticulitermes
spp., Coptotermes spp., Heterotermes spp.) (Mallick et al., 1999).
Although CP is considered only moderately toxic, it is known to possess neurotoxic

and immunotoxic properties and has been shown to be harmful to both animals and humans.
CP has also been reported to cause a reduction in the bacterial, fungal and actinomycete
population of the soil and is known to inhibit nitrogen mineralization in soil. Detection of
CP contamination in surface water bodies and associated sediments has heightened public
concern on the topic and urgent attention and treatment of the problem is recommended.
The environment preservation is one of the aims of the sustainable development. In

India, alarming levels of pesticides have been reported in the air, water, soil as well as in
foods and biological materials. The most important pollutants among the toxicants in India
are organochlorine and organophosphorus pesticides.
The extensive use of pesticides through field application, crop spraying, handling,
rinsing of containers, accidental spills, etc. has a potential to severely contaminate soil.
Most of the pesticides that are in common usage today are known to adversely affect the
functional diversity of the soil microbiota leading to loss of soil fertility and plant growth,
which in turn put the sustainability of agricultural soil at serious risk. To add to the
complexity of the situation, pesticide residues and their metabolites often infiltrate through
the soil surface into the groundwater and cause widespread contamination of aquatic
ecosystems.
The bioremediation is a method that exploits the potential of microbial degradation

for providing a cost-effective and reliable approach to pesticide abatement. Several soil and
aquatic environments have been successfully reclaimed from pesticide contamination by
using microbes capable of degrading the pollutants.
The microbial population adapts to the presence of agrochemicals by change in

species diversity or by adaptation of enzyme systems, so that agrochemicals may be more
rapidly metabolized and consequently eliminated from the environment (Kaufman and
Kearney, 1965). Microbes that are present in pesticide-contaminated sites for a long
duration, develop the ability to degrade or tolerate such contaminants. Such microbes with
advanced traits can be used for pesticide degradation (Farhan et al., 2012). Catabolism and
detoxification metabolisms occur when a soil microorganism uses the pesticide as a carbon
and energy source. Hydrolysis, either chemically or as a result of microbial activity,
degrades CP by converting it to diethyl thio-phosphoric acid (DETP) and 3-5-6-trichloro-
2-pyridinol (TCP).
On the basis of the capacity of microorganisms to adapt to these agrochemicals, it

is possible that they may decontaminate the soil from undesirable chemicals and their
residues. The potential risks associated with the use of this pesticides have provoked the
scientists to find out the ways based on biological and biotechnological approach to
mitigate the ill effects of chlorpyrifos on environmental quality and public health (Harish
et al., 2013).
Pesticide degrading bacteria found in soil are known to have multifarious abilities
such as phytohormone production, mineral solubilization and N2-fixation, etc., which are
extremely crucial for the promotion of plant growth. Presence of the above-mentioned traits
underline and emphasize the agronomic and environmental significance of such microbes.
Considering that chlorpyrifos is one of the most commonly applied insecticides,
keeping in view the human health and ecological risks associated with chlorpyrifos and its
widespread use in the TBP command area, we choose to investigate chlorpyrifos
degradation using biological method, so that possible cost-effective bioremediation
technologies for the environmental clean-up of this toxic pesticide may be developed.
The purpose of this experiment is to isolate and characterize chlorpyrifos

degrading-bacteria, to investigate their degradation potential, to assess their adaptation to
high concentrations of chlorpyrifos and to determine their usefulness in biodegradation of
contaminated sources as well as in improving the growth parameters of rice.
Objectives
1. To isolate and screen efficient chlorpyrifos degrading bacteria from the rhizosphere of
rice
2. To characterize the efficient chlorpyrifos degrading bacterial isolates
3. To evaluate the effect of efficient chlorpyrifos degrading bacterial isolates on the growth
of rice
Review of Literature
II. REVIEW OF LITERATURE
Indiscriminate use of insecticides leads to environmental havoc and poses a great

threat to soil health and beneficial microorganisms. The aim of the present work is to
isolate, screen and characterize efficient chlorpyrifos degrading bacteria and to evaluate
their effect on growth of rice. The review pertaining to isolation, characterization,
biodegradation efficiency and PGPR activity has been documented in the present chapter.
2.1 Isolation and screening the efficient chlorpyrifos degrading bacteria
Brajesh et al. (2003) used liquid enrichment culture to isolate a chlorpyrifos-

degrading bacterium utilizing soil as the source of the inoculum. DNA probing indicated
that genes similar to known organophosphate-degrading (opd) genes were present in the
soils.
Yang et al. (2005) isolated a bacterium from contaminated soils around a chemical
factory and named the strain DSP3. It was capable of biodegrading chlorpyrifos and 3, 5,
6-trichloro-2-pyridinol. Based on the results of phylogenetic similarity, DSP3 was
identified as Alcaligenes faecalis. An addition of strain DSP3 (108 cells g-1) to soil with
chlorpyrifos (100 mg kg-1) resulted in a higher degradation rate than the one obtained from
uninoculated soils.
Diksha et al. (2007) isolated three bacterial strains from pesticide decipitated chilli
crop field soil. The bacterial strains were inoculated in a nutrient media containing
chlorpyrifos, as a carbon source 200 mg/L and incubated at 30 °C for 24 hours in a rotary
shaker. All the strains were named as DSB1, DSB2 and DSB3. Further the degradation of
chlorpyrifos by the three strains was analysed by high performance liquid chromatography
and the degradation by the three strains was compared. The strain DSB1 showed significant
degradation ability in comparison with DSB2 and DSB3 strains, degrading 71.2 % of total
Chlorpyrifos.
Seo et al. (2007) isolated nineteen bacterial strains from petroleum-contaminated

soil in Hilo. They were characterized by two different spray-plated methods, turbidity test
in liquid medium, and 16S rRNA gene sequence analysis. Turbidity tests showed that
strains P1-1, JS14, and JS19b1 utilized several organophosphorus pesticides as growth
substrate. P1-1 can degrade carbofenothion, chlorfenvinphos, diazinon, fonofos, and
pirimiphos-methyl.
Mingfen et al. (2011) isolated a strain of bacteria X1 that can effectively degrade
chlorpyrifos from the activated sludge in the aeration biological tank of municipal sewage
treatment plant. The degradation of chlorpyrifos and the growth are measured by different
carbon and nitrogen sources, pH, and temperature. The results showed that, X1 had good
growth and degradation rates when the pH value was 5.0 and 9.0, the temperature about
30 °C, and the best incubation time was two days. This study determined the best culture
conditions for the bacterial X1 growth and degradation of chlorpyrifos.
Sumitkumar (2011) isolated six bacterial isolates capable of degrading chlorpyrifos

by enrichment from soil samples. The highest value of MIC (100 mg/L) was reported for
bacterial isolate GCC-1. The viable cell count of all six isolates was in the order of 108 per
gram of soil in presence of chlorpyrifos (50 mg/L).
Vijayalakshmi and Usha (2012) isolated the bacteria capable of degrading

chlorpyrifos and Endosulfan. Screening of the isolates for their efficiencies in degrading
chlorpyrifos was carried out based on spectrophotometric analysis. Strains CHS23 and
ENS10 showed the highest degradation of 38 % and 42 %, respectively.
Latifi et al. (2012) isolated several bacterial strains from effluent storage pools of
factories producing pesticides and from soil moisture around them. The isolates were
capable of utilizing chlorpyrifos (CP) as the sole source of carbon, phosphorus and energy.
Isolates were named as IRLM.1, IRLM.2, IRLM.3, IRLM.4, and IRLM.5. IRLM.1 was
able to grow at concentrations of chlorpyrifos up to 2000 mg L-1 and was selected as a
preferable isolate for further analysis.
Wang et al. (2014) isolated a highly effective chlorpyrifos (CP) degrading

bacterium (termed strain X1) from the sludge of drain outlet of a chlorpyrifos manufacturer.
Strain X1 was identified as Cupriavidus taiwanensis based upon the analysis of the 16S
rDNA gene and biochemical characteristics, which was capable of transforming CP into 3-
5-6-trichloro-2-pyridinol (TCP). Degradation experiments were carried out and the
optimized pH and temperature were 7.0 and 32 °C, respectively.
400 mg L-1 of CP was completely transformed within 36 h; approximately 95 % of CP was
removed within 48 h when concentration of CP was up to 500 mg L-1.
Hindumathy and Gayathri (2012) isolated chlorpyrifos degrading bacteria from

rhizospheric and non–rhizospheric soil and the efficiency of microbial consortium for
chlorpyrifos degradation were studied. Maximum 84.5 % dissipation was observed through
bacterial isolate in presence of glucose as compared to 73.3 % dissipation in absence of
glucose. The isolate showed resistance to chlorpyrifos at 10 ppm concentration and also
brought about significant dissipation of this pesticide.
Yan et al. (2012) isolated a fungus capable of using chlorpyrifos as the sole carbon
source was from organophosphate-contaminated soil and characterized as Cladosporium
cladosporioides Hu-01. A novel chlorpyrifos hydrolase from cell extract was purified 35.6-
fold to apparent homogeneity with 38.5 % overall recovery by ammonium sulphate
precipitation, gel filtration chromatography and anion-exchange chromatography.
Fattahi et al. (2012) collected soil samples from rice fields with a history of toxic
pollution. A minimal salt broth medium containing 100 mg/L chlorpyrifos as the carbon
and energy source was used for isolating pesticide-degrading bacteria. Four chlorpyrifos-
degrading bacterial strains were isolated from the soils which include Bacillus
licheniformis strain IARI-M-12, Bacillus pumilus strain MS42, Bacillus cereus strain
ESB15 and Delftia tsuruhatensis strain SJ113.
Niti et al. (2013) obtained 26 bacterial isolates from chlorpyrifos contaminated soil
samples by enrichment culture technique. Out of these, four isolates showed growth up to
30,000 ppm and four isolates up to 40,000 ppm chlorpyrifos amended in mineral salt
medium (MSM) containing glucose (0.2 %). Bacterial count was found to be more in the
medium amended with 100 ppm with all the four isolates at 7 days of growth. Maximum
utilization of chlorpyrifos was observed with the isolate SB1 (80.1 %) followed by HIC2
(76.2 %), SGB2 (65.2 %) and HIIGA2 (58.1 %) in liquid medium.
Parmar et al. (2014) isolated nineteen chlorpyrifos degrading bacteria from an

organophosphate pesticides manufacturing area of Gujarat. Biodegradation study of
chlorpyrifos was performed using High Performance Liquid Chromatography (HPLC)
using 300 ppm chlorpyrifos concentration. The most promising chlorpyrifos degrading
bacteria were CPD-1, CPD-4, CPD-9, CPD-15 and CPD-18 which degraded 70 %, 71 %,
66 %, 80 % and 83 %, respectively within 24 hr.
Ehab et al. (2014) isolated a bacterial strain Y242 from agricultural wastewater and
it was found to be highly effective in degrading chlorpyrifos. On the basis of phylogenetic
analysis of 16S rRNA sequence, the isolate was identified as Bacillus subtilis. The
efficiency of the B. subtilis Y242 isolate as a chlorpyrifos degrader was examined under
different culture conditions formulated. It was observed that B. subtilis Y242 was able to
utilize chlorpyrifos as a sole carbon and energy source and grows on a medium containing
concentration up to 150 mg L-1 and recorded 95.12 % pesticide decomposition within 48 h.
Shinde et al. (2015) isolated the organisms from the soil which showed degradation
of the pesticides. 96 isolates were obtained on minimal medium which contained pesticide
as a sole source of carbon. These isolates include both bacteria as well as fungi. Later,
different pesticide concentrations were tested, four isolates were found to efficiently
degrade maximum up to 19 gm percent of pesticide.
Kolengaden et al. (2015) isolated microorganisms capable of degrading

chlorpyrifos from cardamom plantation soil samples by enrichment culture technique.
Finally, 1 ml was plated on MS media containing 100 ppm chlorpyrifos. A fungal isolate
(M5) was obtained which was identified as Isaria farinosa.
Ifediegwu et al. (2015) isolated chlorpyrifos degrading bacteria and identified them
as strains of Pseudomonas aeruginosa, Serretia marcescens and Klebsiella oxytoca. Their
growth in mineral salt medium supplemented with 20 mg L-1 of chlorpyrifos was monitored
at optical density of 600 nm. HPLC analysis of the residual chlorpyrifos after 14 days
incubation showed that Pseudomonas aeruginosa was able to degrade 60 % of the
pesticide; Klebsiella oxytoca degraded 54 %; while, Serretia marcescens had 53 %
reduction of the pesticide concentration in the mineral salt medium.
Rashmi and Dayana (2015) isolated twelve pesticide resistant bacterial isolates
capable of tolerating four pesticides such as chlorpyrifos, malathion, chlordane and
chlorothalonil by spot assay enrichment technique from field soil. Among them, Klebsiella
spp (K1, K2 and K4), Pseudomonas sp (P3) and Bacillus sp (B4) showed more resistance
and were used for further studies. They found that among those organisms, the three
Klebsiella spp were the most effective pesticide tolerating organisms and also found that
the pesticide tolerating property was plasmid origin.
Amareshwari et al. (2015) studied on identification of a novel species of

Flavobacterium bacteria capable to degrade the organophosphorus pesticides. The
bacterium was isolated from agricultural soil collected from Guntur District, Andhra
Pradesh, India. The samples were serially diluted and the aliquots were incubated for a
suitable time following which the suspected colony was subjected to 16S rDNA
sequencing. The sequence thus obtained was aligned pairwise against Flavobacterium
species.
Deepti and Mehvish (2015) isolated two chlorpyrifos degrading bacteria using
serial dilution technique followed by selective enrichment on minimal medium with
chlorpyrifos as the sole carbon source, from soil samples collected from a sugarcane field.
Microbial growth during the study was monitored by measuring the optical density at 620
nm using a spectrophotometer. The best result for growth of both the isolates was observed
on minimal medium enriched with 10 ppm chlorpyrifos at temperature 37 °C incubated for
48 hrs at 150 rpm.
Gireesh et al. (2016) isolated eight bacterial isolates from vegetable crops like
tomato and brinjal by the enriched MSM broth with a supplement of chlorpyrifos source.
Among the eight isolates, CDB-1 isolate utilized the pesticide (chlorpyrifos) effectively
and showed maximum growth bacterial count 5.8 × 106 CFU m L-1. Efficient CDB-1 isolate
and chlorpyrifos degrading capacity were determined using gas chromatographic method.
Sharma et al. (2016) isolated bacterial species from chlorpyrifos contaminated

soils. These were identified as Bacillus and Micrococcus spp by using biochemical tests.
The species were tested for their capabilities to degrade the pesticide at two different
concentrations (0.05 % and 0.1 %) spectrophotometrically. After 10 days of incubation
maximum degradation of chlorpyrifos was observed in Micrococcus sp. with 0.1 % of
pesticide.
Truong and Duong (2016) studied on isolation of the bacterial strains which degrade
chlorpyrifos from rice-upland crop soils in the Mekong Delta. The soil bacteria were
enriched in mineral salt medium containing 20 mg L-1 of chlorpyrifos as the only carbon
source for bacterial growth. The results showed that one bacterial community was enriched
and degraded chlorpyrifos. One bacterial strain (coded as BT-C8.9) that was isolated from
this community degraded 41.07 % of chlorpyrifos after 30 culture days. According to the
sequencing of 16S rRNA gene, this bacterial strain was identified as Microbacterium sp.
C8.9.
Sonali and Tushar (2017) isolated two bacterial strains capable of utilizing
chlorpyrifos as a sole source of carbon and energy for growth viz., TB251 and TB252. The
two strains showed different results for the biochemical tests suggesting genetic diversity
of the population.
Hamsavathani et al. (2017) isolated two pesticide degrading bacteria from

chlorpyrifos contaminated soil by enrichment culture technique and were identified as
Kocuria kristinae and Staphylococcus aureus. The growth response and degradation of
chlorpyrifos by the isolates in MSM broth supplemented with 0.5 % chlorpyrifos was
monitored every 48-72 hrs in spectrophotometer at 600 nm. Kocuria sp showed maximum
growth in 7 days than Staphylococcus aureus. The isolate, S. aureus was more potent in
degrading the 80 % of the total compound from the media in 2 weeks of incubation than K.
kristinae, which shows 35 % of degradation. They concluded that the isolate
Staphylococcus aureus was more potent in degrading chlorpyrifos in liquid culture and can
also be used in bioremediation of chlorpyrifos contaminated soils.
Rayu et al. (2017) studied on sequential soil and liquid culture enrichments that
enabled the isolation of chlorpyrifos degraders such as Xanthomonas sp. 4R3-
M1, Pseudomonas sp. 4H1-M3, and Rhizobium sp. 4H1-M1 was further investigated for
biodegradation of CP and its primary metabolic product, 3-5-6-trichloro-2-pyridinol (TCP).
The results indicate that all three bacterial strains almost completely metabolized CP (10
mg/L) and TCP, occurring as a metabolic degradation product, in mineral salt media as a
sole source of carbon and nitrogen. Xanthomonas sp. 4R3-M1 and Pseudomonas sp. 4H1-
M3 could also degrade TCP (10 mg/L) as a sole carbon and nitrogen source, when provided
externally.
Mirenga et al. (2018) isolated chlorpyrifos and diuron- degrading bacteria from
pesticide exposed agricultural soils in the Nzoia river drainage basin. One soil isolate was
found capable of degrading chlorpyrifos and another was found capable of degrading
Diuron. Sequence analysis of the two isolates using BLASTN and phylogenetic analysis
revealed that the isolate capable of utilizing chlorpyrifos as the sole carbon source was
Kosakonia oryzae strain Ola 51, while the isolate capable of utilizing diuron as the sole
carbon source was Pseudomonas aeruginosa strain M1.
2.2 Characterization of the efficient chlorpyrifos degrading bacterial isolates
Xiaohui et al. (2007) isolated a highly effective chlorpyrifos-degrading bacterium

strain Dsp-2 from the polluted treatment system of a chlorpyrifos manufacturer. This strain
was preliminarily identified as Sphingomonas sp. based on its morphological, physiological
and biochemical tests as well as 16S rDNA analysis. It utilized chlorpyrifos as its sole
source of carbon for growth, by hydrolysing chlorpyrifos to 3-5-6-trichloro-2-pyridinol
(TCP).
Ghanem et al. (2007) isolated a chlorpyrifos (CP)-degrading bacterial strain

Klebsiella sp. from an activated sludge sample collected from the Damascus waste-water
treatment plant and the identification was done using major staining and biochemical
differentiation tests. Within 4 days of incubation, the isolated Klebsiella sp. was found to
break down 92 % of CP when co-incubated in a poor mineral medium in which CP was the
sole carbon source (13.9 g L-1 poor medium).
Rani et al. (2008) isolated a soil bacterium capable of utilizing chlorpyrifos as sole
carbon source by selective enrichment on mineral medium containing chlorpyrifos. The
bacterial isolate designated as MS09, was identified and characterized as a strain of
Providencia stuartii based on biochemical characteristics and 16S rRNA sequence analysis.
Growth studies showed that P. stuartii strain MS09 utilized chlorpyrifos to grow in Luria-
Bertani broth containing different concentrations of chlorpyrifos at 50-700 mg/L.
Sayali et al. (2012) used enrichment culture technique to isolate bacterial strains
from garden soil. The five pure bacterial cultures, named EC1, EC2, EC3, EC4 and EC5
were isolated and subsequently characterized by 16S rRNA gene sequencing and
biochemical tests. The bacterial isolates were able to grow in medium containing the
individual pesticide as the carbon source.
Das and Adhya (2012) isolated a soil bacterium capable of utilizing chlorpyrifos as
the sole source of carbon and energy from rice soil enriched with repeated application of
chlorpyrifos (10 mgkg-1). The strain named CRRI NF3, was preliminarily identified as
Bacillus sp. based on its morphological, physiological and biochemical tests as well as 16s
rRNA gene sequence analysis.
Savitha and Saraswathi (2012) worked on isolation, identification and

characterization of the chlorpyrifos resistant bacterial isolates from cabbage cultivated soil
of a private agricultural farm in Bangalore, India. Fifteen bacterial isolates capable of
degrading chlorpyrifos were isolated. Out of them, three chlorpyrifos hyper resistant
bacteria were finally selected for follow up studies. Three isolates were identified as
Bacillus stearothermophilus, Bacillus circulans and Bacillus macerans.
Gireesh et al. (2016) isolated sixteen bacterial isolates from insecticide treated soils
by enriching mineral salt medium broth with supplement of chlorpyrifos, Phorate source.
These isolates were characterized on the basis of cell morphology, cultural and biochemical
properties. Among the eight chlorpyrifos degrading bacterial isolates, CDB-1 isolate
utilized the more pesticide and was identified as Pseudomonas sp.
Supreeth and Raju (2016) isolated bacterial isolate capable of mineralizing

chlorpyrifos without accumulation of TCP from agricultural soil by enrichment method.
Based on morphological, biochemical characterization and with Bergey’s Manual
comparison, the isolate was identified as Staphylococcus sp. The isolate was found to
metabolize chlorpyrifos completely in mineral salt medium with chlorpyrifos as the sole
carbon source.
Salem et al. (2016) isolated, screened and characterized bacteria which tolerates the
two pesticides compounds (chlorpyrifos and dimethoate). Bacterial strains that could
persist the presence of the both organophosphorus compounds were isolated and identified
by biochemical test. The obtained results from pesticides enriched cultures showed the
presence of Enterobacter cloacae, and Panteoa sp.
Sunitha and Onkarappa (2018) focused on isolation of actinomycetes from coffee

plantation soil, degradation of chlorpyrifos and their residual analysis by GC-MS. Total 29
isolates were recovered and subjected for morphological and biochemical characterization
studies, isolates are belonging to Streptomyces species. Biodegradation of pesticides were
detected by using chlorpyrifos as sole source of carbon in different concentrations and
further bulk cultured with potent isolates to detect residues by GC-MS
2.3 In vitro screening of the bacterial isolates for their beneficial attributes
Supraja et al. (2011) isolated fifteen fluorescent Pseudomonas isolates from

rhizospheric soils of Red gram and Maize crops in the Rangareddy district, Telangana.
These test isolates were biochemically characterized and screened in vitro for their plant
growth promoting traits like phosphate solubilization, production of Indole Acetic Acid
(IAA), Hydrogen Cyanide (HCN) and siderophore. Due to production of HCN and
siderophores, fluorescent Pseudomonas isolates inhibited the growth of Fusarium
moniliforme.
Rhizobacteria were found in the rhizosphere of plants and are beneficial for plant
development. They promote plant growth by increasing the availability of nutrients,
increasing the uptake of nitrogen, phosphorus etc. described by Ram and Singh (2012).
PGPR help plants directly by secreting phytohormones and some other plant growth-
promoting substances. PGPR also protects plants against pathogens by exploiting their
antagonistic potential. Some species of PGPR help plants by exploring root growth for their
stability in stressed conditions.
Pankaj et al. (2012) isolated seven bacterial isolates from rhizosphere of common
bean growing at Uttarakhand Himalaya and screened them for their potential plant growth
promoting (PGP) and antagonistic activities. Strain BPR7 produced IAA, siderophore,
phytase, organic acid, ACC deaminase, cyanogens, lytic enzymes, oxalate oxidase and
solubilized various sources of organic and inorganic phosphates.
Bhattacharyya and Jha (2012) reviewed on variety of applications related to

agricultural improvement along with their mechanism of action with special reference to
plant growth promoting traits are summarized.
Sarvani and Reddy (2012) isolated thirty Bacillus cultures from the rhizospheric
soils of Groundnut and Redgram crops growing in Rangareddy district, India and identified
based on biochemical characteristics. These were screened for PGP attributes and
antagonism against soil borne phytopathogens viz., Sclerotium rolfsii, Rhizoctonia solani
and Fusarium solani. Results revealed that 50 % (15/30 isolates) reacted positively for one
or more PGP properties.
Muhammad and Muhammad (2013) reviewed on PGPR as a direct and indirect tool
to improve the production of crops by various mechanisms like production of auxins,
siderophores, HCN and P- solubilization, etc., by different genera of rhizospheric bacteria
such as Azospirillium, Azotobactor, Pseudomonas, Bacillus, Enterobactor and Rhizobium
etc.
Ajaykumar et al. (2014) collected six French bean rhizospheric soil sample isolates
from different locations of Shimla and Solan in H.P (India). A total of 30 bacteria were
isolated and screened for different plant growth promotion properties i.e. phosphate
solubilization, IAA production, Ammonia production, ACC deaminase activity, HCN
production and catalase. Results showed that 12 bacterial isolates were positive for
phosphate solubilization. IAA production was shown by almost all the bacterial isolates.
Three isolates were positive for ammonia production. ACC deaminase activity was shown
by nine isolates. Two isolates were positive for HCN production and all the isolates were
found to be catalase positive.
2.3.1 IAA production
Sadaf et al. (2009) studied the indole acetic acid production and enhanced
plant growth promotion by indigenous PSBs. Auxin production by PSB bacteria was
determined via bioassay and HPLC by the bacteria in liquid culture. Indole acetic acid and
Indole butyric acid were produced by these bacteria in varying concentration with and
without the addition of tryptophan. Three promising isolates CMG854, CMG857 and
CMG860 were investigated to establish the effect on plant growth.
Madhuri (2011) isolated and screened the bacterial isolates from leguminous plants
groundnut, chickpea, fenugreek and lucerne. Results revealed that, out of 60 colonies
selected from rhizosphere of groundnut, 40 colonies from rhizosphere of groundnut and 20
colonies from rhizosphere of Lucerne showed red color with Salkowaski’s reagent on nylon
6’6’ membrane indicating production of IAA by the organisms.
Mohite (2013) isolated characterized and identified the indole acetic acid producing
bacteria from the rhizospheric soil. Out of ten Indole acetic acid producing isolates, five
were selected as efficient producers. Among the isolates BR3 and WR2 were found to be
the best producer of IAA. On the other hand, BR1, BR2 and MR2 were found to be a
medium producer of IAA.
Jeyanthi and Ganesh (2013) isolated Pseudomonas fluorescence from rhizosphere

soil and studied the production of indole acetic acid utilization. The effect of
L-tryptophan was studied and the highest yield of 58 μg ml-1 at 72 h was obtained by using
0.5 mg ml-1 concentration. The best yield of 53 μg ml-1 was obtained with medium
supplemented with 1 % lactose. The maximum yield of 48 μg ml-1 was obtained with
0.2 % supplemented peptone as an organic nitrogen source.
Reetha et al. (2014) isolated Pseudomonas fluorescens and Bacillus subtilis from
rhizosphere of onion and analyzed these bacteria under in vitro conditions for indole acetic
acid production and studied the effect of these bacteria on plant growth of onion plant.
Results showed that both bacteria increased the root length, shoot length, root and shoot
fresh and dry weight and onion seeds over control.
Dweipayan et al. (2015) developed a simple, quick and reliable method for the
detection and quantitation of indole acetic acid (IAA) and indole-3-butyrate (IBA), an auxin
phytohormone produced by rhizobacteria from L-tryptophan (Trp) metabolism using High
Performance Thin-Layer Chromatography (HPTLC). Results revealed that the ability of
Pseudomonas putida strain A (AB667903) to produce Indole-3 Lactic Acid (ILA), IAA
and Indole-3 Acetamide (IAM) from Trp, qualitative analysis showed that ILA was most
abundantly produced (2.6–34.0 μg ml-1), followed by IAA (0.7–10.3 μg ml-1)
2.3.2 Siderophore production
Anand and Kulothungan (2010) isolated Pseudomonas fluorescens from groundnut

rhizosphere and tested for their ability to produce iron chelating siderophores using
standard protocols. Maximum production of siderophores was found to be 4.92
μ mol benzoic acid ml-1.
Tailor and Joshi (2012) isolated seven bacterial isolates from sugarcane
rhizosphere. All the isolates were found to produce more than 85 % siderophore units.
Among them S-11 was found be the most efficient siderophore producer (96 % SU). S-11
was further characterized and identified as Pseudomonas fluorescens.
Sreedevi et al. (2014) isolated ten Pseudomonas spp. from Paddy soil. Among
isolated strains, three Pseudomonas isolates P1, P2 and P3 were shown siderophore
production on Succinic acid medium and Chromo-azural S agar plate medium. Maximum
siderophore production was 94, 88 and 83 % for Pseudomonas 1, Pseudomonas 2 and
Pseudomonas 3 isolates, respectively. All three isolates have shown both type of
siderophore production i.e., wine red colour formation in supernatant indicated production
of hydroxamate type (pyoverdine), while yellow colour formation in supernatant showed
presence of catecholate or phenolate type (pyochelin) siderophore.
Sabrina et al. (2014) described the synthesis and the biological properties (Fe
uptake, binding to FptA) of several thiazole analogues of pyochelin. Among them the two
first pyochelin analogues able to bind FptA without promoting any iron uptake in P.
aeruginosa.
Bruno et al. (2015) studied the contribution of siderophore mediated iron uptake
and storage to M. robertsii fitness and strong up regulation of the insect iron-binding
proteins, transferrins, during infection. Insect bioassays revealed that, ferricrocin is
required for full virulence against Spodoptera exigua.
Veronique et al. (2015) studied the detailed process of Pyoverdine I (PVDI) and
pyochelin (PCH) siderophores produced by Pseudomonas aeruginosa PAO1 and the
cytoplasmic enzymes involved in each of these two siderophore biosynthesis pathways can
form siderophore-specific multi-enzymatic complexes called siderosomes associated with
the inner leaflet of the cytoplasmic membrane has discussed.
2.3.3 P- Solubilisation
Eighty Pseudomonas strains were screened for phosphate solubilization. Out of 80,
three isolates (P. aeruginosa, P. plecoglossicida and P. mosselii) showed the ability to
solubilize tri-calcium phosphate by Jha et al. (2009). They reported that, P. plecoglossicida
and P. mosselii can be used as biofertilizers because of the innate potential of phosphate
solubilization.
Oliveira et al. (2009) reported several phosphate solubilizing microorganisms from

Maize rhizospheric soil. The strains of bacteria, fungi and most of the actinobacteria were
confirmed as PSM with high ‘P’ solubilization activity. Among the different bacterial
isolates, B17 and B5 were identified as Bacillus sp. (B17) and Burkholderia sp. (B5),
respectively. The most efficient ‘P’ solubilizing strains were from P-Ca source culture
solution. The solubilization was 67 % and 58.5 % of the total ‘P’, respectively. The B5
isolate was recorded the highest reduction in pH (4.46) in the growth solution.
Banerjee et al. (2010) isolated two stress tolerant phosphate solubilizing

rhizobacteria, Arthrobacter spp. and Bacillus spp. from tomato rhizosphere and
characterized using morphological and biochemical tests. In addition to phosphate
solubilizing ability these strains were also studied for various plant growth promotion and
biocontrol activities including indole acetic acid (IAA) production.
Keneni et al. (2010) reported the phosphate solubilization efficiency of the five
isolates along with Jim-41 using different P sources [tricalcium phosphate (TCP), Egyptian
Rock Phosphate (ERP), Bikilal Rock Phosphate (BRP) and Old Bone meal (OB)]. The PSB
isolates were significantly solubilized a higher amount of TCP, ERP and OB over the
uninoculated control.
Manivannan et al. (2011) reported six different Phosphate solubilizing

microorganisms (Pseudomonas sp., Bacillus sp., Penicillium sp. and 3 strains of
Aspergillus spp.) from the rhizosphere soils of chilli and tomato plants. They found that
Penicillium sp. solubilized tricalcium phosphate up to 1045 μgml-1 and Pseudomonas sp.
up to 985 μg ml-1 after 14 days of incubation in Pikovskaya's broth
Mahantesh and Patil (2011) isolated phosphate solubilizing bacteria from the area
around Bidar region and screened on the basis of their solubilization of inorganic tricalcium
phosphate in liquid broth. Ten strains that had higher solubilization potential were selected
and characterized.
Uma and Sathiyavani (2012) reported solubilization of phosphate by Bacillus spp,

from groundnut rhizosphere soil (Arachis hypogaea L). The isolated strains were confirmed
as B. subtilis and B. cereus. The effect of different pH, temperature, carbon sources,
nitrogen and potash of the phosphate solubilization was optimized. Maximum
solubilization was recorded at temperature 40 °C.
Hettiarachchi et al. (2013) evaluated the ability of phosphorus solubilizing potential

of bacterial and fungal isolates from Hevea rhizosphere and their effective biofilm
communities were evaluated using solid and liquid media under in vitro conditions. Results
revealed that the biofilm showed significantly higher P-solubilization (853.3 ± 25.17 mg l-
1
) than their bacterial and fungal individual cultures.
Fekadu (2013) isolated twelve P. fluorescens strains from rhizospheric soil of Faba
bean and tested for phosphate solubilization. All tested isolates of P. fluorescens strains
have a potential of phosphate solubilization on Pikovskaya’s media. It was summarized
that all P. fluorescens strains can be used as bio-fertilizers for soil fertility improvement.
Zhiguang et al. (2015) isolated twenty phosphate-solubilizing bacteria (PSB) from

calcareous rhizosphere soils. These bacterial strains were identified as species of B.
megaterium (B. aryabhattai), B. subtilis, P. aeruginosa, Rhizobium spp., Acinetobacter spp
and Pseudomonas oryzihabitans using 16S rRNA analysis. Results showed that halo zone
formation by PSB on NBRIP plates was a good indicator. The NBRIP liquid culture showed
that four PSB strains lowered medium pH (<4.3) and released WS-P up to 523.69
mg L-1 within three days incubation.
Ida et al. (2015) conducted an experiment to determine the potency of phosphate

solubilizing microbes (PSM) and studied the solubilization of P and P uptake of plant in
peat soil. Based on their ribosomal DNA, the best PSM were identified as Burkholderia
gladioli and Penicillium aculeatum that yielded the highest growth and maximum uptake
of a phosphate by oil palm seedling.
Manoharan et al. (2016) isolated two salt tolerant endophytic and phosphate
solubilizing bacteria ACMS25 and PVMX4 from Phyllanthus amarus. They were
identified as Acinetobacter sp. and Bacillus sp. based on 16s rRNA sequencing. Both the
strains were found to be positive for most of plant growth promoting traits. Under in vitro
conditions at 160 mM NaCl, both the endophytes alone or in combination promoted a
higher vigour index, germination (%), plant biomass, P content, plant phenolic content,
radical scavenging and antioxidative activity, compared to the standard strain Bacillus
megaterium MTCC446 and un-inoculated control.
Ranjan et al. (2016) screened three potent phosphate solubilizing bacterial strains
(P4, P9 and P10) associated with L. cernuum rhizoids and identified by 16S rDNA
homologies on Ez-Taxon database as Burkholderia tropica, B. unamae and B. cepacia,
respectively. Day wise kinetics of phosphate solubilization against Ca3 (PO4)2 suggested
P4 (580.56 ± 13.38 g ml−1) as maximum mineral phosphate solubilizer followed by P9
(517.12 ± 17.15 g ml−1) and P10 (485.18 ± 14.23 g ml−1).
Manouchehr et al. (2016) studied the phosphate solubilization potential of

inhabiting bacteria from rhizosphere of Avicennia marina plants. Among 13 isolates
screened, four high phosphate solubilizing bacteria were selected based on the formation
and dimension of clear halo around the colonies. Phylogenetic analysis of 16S rDNA of
PSBs showed their close similarity to Bacillus, Pseudomonas and Acinetobacter species.
Results showed that PSB1 and PSB3 were found as the most potent isolates with 3.5 and
2.6 Phosphate Solubilization Index (PSI) values, respectively. P solubilizing efficiency was
also achieved in a high percentage range for consortium culture (74-78 %) and PSB10 (71-
77 %).
2.4. Biodegradation analysis of the chlorpyrifos degrading bacterial strains
A bacterial strain C2A1 was isolated by Anwar et al. (2009) from soil, which was
found to be highly effective in degrading chlorpyrifos and its first hydrolysis metabolite
3,5,6-trichloro-2-pyridinol (TCP). Strain C2A1 was identified as Bacillus pumilus. Strain
C2A1 utilized chlorpyrifos as the sole source of carbon and energy. At high pH 8.5 and
high inoculum density when chlorpyrifos was used as the sole source of carbon and energy,
maximum pesticide degradation was observed. Within 8 days of incubation the strain C2A1
showed 90 % degradation of TCP (300 mg/L).
By selective enrichment technique, Jayamadhuri and Ramaswamy (2009) isolated

Bacillus spp. and Pseudomonas spp. These isolates were tested for their ability to degrade
the respective insecticides in mineral salts medium. Nearly 75 % of chlorpyrifos and
phorate and 50 % of dichlorvos, methyl parathion and methomyl were degraded by cultures
of soil bacteria within 7 days of incubation. Formation of one unidentified metabolite in
inoculated samples were revealed by qualitative analysis of chlorpyrifos and methyl
parathion residues by gas chromatography.
Vidyalakshmi et al. (2009) developed three aerobic bacterial consortia, AC, BC and
DC, from pesticide-contaminated soils of Punjab and they were able to degrade
chlorpyrifos after 21 days of incubation in basal medium by 54 %, 46 %, and 61 % and
chlorpyrifos (50 mg/L) in soil after 30 days by 50 %, 56 % and 64 %. P. aeruginosa, B.
cereus, Klebsiella sp., and Serratia marscecens obtained from these consortia showed
92 %, 60 %, 56 % and 37 % degradation of chlorpyrifos (50 mg/L) in soil after 30 days.
A strain ZHU-1 capable of utilizing Chlorpyrifos as the sole carbon sources and
energy was isolated by Zhu et al. (2010) from soil. Based on analysis of morphology,
physiological and biochemical characters and 16S rRNA ZHU-1 was identified as Bacillus
licheniformis. The addition of ZHU-1 to soil treated with chlorpyrifos resulted in a higher
degradation rate than uninoculated soils, the degradation rate of chlorpyrifos (100 mg kg-
1
) could reach 99 % or above after 14 days. The microbial manure added by strain ZHU-1
could be applied not only as fertilizer, but also in degrading chlorpyrifos residue in soil.
Singh et al. (2011) studied chlorpyrifos degradation by a rice field cyanobacterium

Synechocystis sp. strain PUPCCC 64 so that the organism is able to reduce insecticide
pollution in situ. The unicellular cyanobacterium was isolated and identified by partial 16S
rRNA gene sequence. The tolerance limit of the organism was determined by studying its
growth in degraded concentrations (2.5-20 mg/L) of chlorpyrifos. The degradation products
were evidenced by GC-MS chromatogram. One of the degradation products was identified
as 3-5-6 trichloro-2-pyridinol.
Sumitkumar (2011) investigated the chlorpyrifos degrading capability of four

bacterial monocultures (RCC-2, GCC-1, GCC-3 and JCC-3) and two bacterial mixed-
cultures (GCE345 and GCC134) in terms of treatment duration and culture volume, using
soil slurry medium. Among the bacterial monocultures, RCC-2 was found to be most
efficient with 21, 37, 54 and 77 % of chlorpyrifos degradation in 5, 10, 15 and 30 days of
treatment duration, respectively. Out of two bacterial mixed-cultures, GCC134 was more
effective and resulted in 24, 38, 56 and 85 % degradation of chlorpyrifos in 5, 10, 15 and
30 days of treatment, respectively. Chlorpyrifos degradation was higher by increasing the
culture volume of respective bacterial cultures, from 10 % to 25 %, for 10 days of treatment
at room temperature.
Maya et al. (2011) investigated the efficacy of soil bacterial communities

comprising seven different isolates for biodegradation of chlorpyrifos and TCP, a
degradation product of chlorpyrifos. Chlorpyrifos concentration ranged from 25 to 200 mg
L-1 and that of TCP ranged from 25-100 mg L-1. The genetic relatedness of the isolates 1-4
with Pseudomonas spp., isolates 5 and 6 with Agrobacterium spp. and isolate 7 with
Bacillus spp. was confirmed with 16s rRNA gene sequence analysis. Their degradation
potential for chlorpyrifos and TCP was found to be in the order
Pseudomonas>Agrobacterium>Bacillus.
Pseudomonas spp. from industrial drain was isolated by Farhan et al. (2012). Good
degradation efficiency was shown by this strain as compared to the control. Complete
biodegradation yielded carbon source and energy by the process of oxidation which was
used for the growth of microbes.
The combined use of plants and microbes for chlorpyrifos biodegradation was
investigated by Ahmad et al. (2012). The plant and microbes used were rye grass and
Bacillus pumilis. The combination of plant and microbes degraded 97 % of chlorpyrifos
within 45 days in soil experimentation. The fact that exogenous microbes could be used
successfully for the bioremediation process was highlighted by this study.
Chitrambalam et al. (2012) isolated four bacterial isolates namely Pseudomonas
putida (NII 1117), Klebsiella sp., (NII 1118), Pseudomonas stutzeri (NII 1119),
P. aeruginosa (NII 1120) present in the consortia were identified on the basis of 16S rDNA
analysis. The degradation studies were carried out at neutral pH and temperature 37 °C with
chlorpyrifos concentration 500 mg L-1. LC-mass spectral analysis showed the presence of
metabolites chlorpyrifos-oxon and Diethyl-phosphorothioate. These results highlight an
important potential use of this consortium for the clean-up of chlorpyrifos contaminated
sites.
Nagavardhanam and Vishnuvardhan (2013) carried out bioremediation of the

chlorpyrifos using pure culture of Kocuria sp. and the cloned culture in a shake flask under
controlled environmental conditions. The GC data showed that chlorpyrifos was degraded
up to 52 % in the cloned culture containing 3.84 g L-1 chlorpyrifos while in native Kocuria
sp. containing 3.84 g L-1 chlorpyrifos, it was degraded up to 75 %. Thus, the microorganism
Kocuria sp. and the cloned culture were found to be well adapted to chlorpyrifos.
Rokade and Mali (2013) described the biodegradation of chlorpyrifos by

P. desmolyticum NCIM 2112. The pH and temperature optima for degradation were found
to be 7.0 and at 30 °C respectively. Biodegradation was influenced by other carbon and
nitrogen sources and indicated that glucose and maltose were effective at 0.5 %
concentration and sodium nitrate and yeast extract at 0.05 %. P. desmolyticum NCIM 2112
degraded chlorpyrifos into nontoxic metabolites like 2-pyridinol and thiophosphate.
Lu et al. (2013) isolated and characterized a bacterial strain, Cupriavidus spp.

DT-1 which was capable of degrading chlorpyrifos and 3, 5, 6-trichloro-2-pyridinol (TCP).
These compounds were used as sole carbon source. The degradation pathway was
investigated and it was shown that chlorpyrifos was first hydrolysed to TCP, successively
dechlorinated to 2-pyridinol and then subjected to the cleavage of the pyridine ring and
further degradation. Chlorpyrifos-contaminated soil was inoculated with strain DT-1 which
resulted in a degradation rate of chlorpyrifos and TCP of 100 % and 94.3 %, respectively
as compared to a rate of 28.2 % and 19.9 % in uninoculated soil.
Barathidasan et al. (2014) obtained a chlorpyrifos degrading bacterial strain

(Cellulomonas fimi) and a TCP utilizing fungal strain (Phanerochaete chrysosporium) from
Microbial Type Culture Collection (MTCC), Chandigarh. The fungus could also degrade
50 mg chlorpyrifos L-1 within 6 days. Co-culture completely mineralized 50 mg
chlorpyrifos L-1 within 16 h at 33 ºC and at pH 8.4.
Anish et al. (2016) isolated a bacterial strain AST-2.2 with chlorpyrifos degrading
ability by enrichment technique from apple orchard soil with previous history of
chlorpyrifos use. The strain AST-2.2 utilized chlorpyrifos as the sole source of carbon and
energy. This strain exhibited growth up to 400 mg L-1 concentration of chlorpyrifos and
exhibited high extracellular Organo-Phosphorus Hydrolase (OPH) activity. Gas
Chromatography-Flame Ionization Detector (GC-FID) studies revealed that P.
resinovarans AST2.2 degraded 43.90 % of chlorpyrifos (400 mg L-1) within 96 hrs.
Intermediates of chlorpyrifos degradation were identified using GC-MS.
2.5 Evaluation of the effect of efficient pesticide degrading bacterial isolates under in
vivo conditions
Akbar and Sikander (2016) isolated two bacterial strains, JCp4 and FCp1,
exhibiting chlorpyrifos-degradation potential from pesticide contaminated agricultural
fields. Influence of bacterial presence on plant growth and pesticide degradation was
studied using plant growth experiments taking cowpea as test crop. Cp addition to soil
affected a reduction in certain plant parameters such as % germination (14.2%), shoot
length (2 cm), shoot fresh weight (0.29 g), root fresh weight (0.05 g), shoot dry weight (0.2
g) and root dry weight (0.02 g). Plants grown in CP supplemented soils inoculated with CP-
degrading bacterial strains exhibited significant enhancement in growth in terms of height
and weight.
Rajkumar et al. (2014) tested the effects of technical grade hexachlorocyclohexane

(tech-HCH) on the germination of radish and green gram seeds. Protease and amylase
activities were reduced in seeds grown in soil spiked with tech-HCH. The degradation of
25 μg tech-HCH g−1 soil was complete by 120 h. The seed germination and the activities
of the assayed enzymes were better in bioremediated soils.
Andriani et al. (2017) used the bacteria Enterobacter cloacae, Enterobacter sp and
P. fluorescens which potentially degraded glyphosate and showed comparatively higher
germination percentage, root length and shoot length of paddy during the investigation. A
positive result indicates that bacterial growth boosters from the plant (endophyte) as well
as the area of rooting (rhizosphere) have additional potential as biofertilizer, bio stimulant
and bio protectant but also as bio-degraders of pollutants such as the herbicide glyphosate.
Bhagat et al. (2017) isolated six pesticide (chlorpyrifos) degrading bacteria were
isolated from pesticide polluted soil samples. A pot assay was also conducted to check the
efficacy of the two best isolates for pesticide degradation and growth of Capsicum annuum
plant. The pesticide degradation by these two isolates was confirmed using gas
chromatography technique and it was observed that there was significant difference
between the chlorpyrifos degrading inoculants over the control. The growth parameters
revealed that there was significant difference in the inoculated treatments compared to
control.
Material and Methods
III. MATERIAL AND METHODS
The present investigation on the chlorpyrifos degrading bacteria in rice ecosystem

was carried out during 2017-2018. The laboratory and pot experiments were carried out in
the Department of Agricultural Microbiology and Pesticide Residue and Food Quality
Analysis Laboratory, University of Agricultural Sciences, Raichur, which is situated in the
Northeastern dry zone (Zone-2) of Karnataka (at 16⁰ 12’ N latitude, 77⁰ 21’ E longitude
and at an altitude of 389.37 m above mean sea level). The details of the material used and
the methodology followed is described in this chapter.
3.1 Collection of soil samples for the isolation of chlorpyrifos degrading bacteria
A survey was conducted for selection of various sites of rice growing region TBP
command area of Hyderabad Karnataka. Raichur and Koppal districts are the two major
rice growing districts, which were selected for the present study. Fourteen geographical
locations of these two districts were finalized with a minimum of 10 km distance between
them. Soil samples were collected from the top 10 to 15 cm of the soil profile in the paddy
rhizosphere and they were carried in sterilized polythene bags. The polythene bags were
properly tied; labeled and at most care was taken to avoid contamination. The soil samples
were preserved in a refrigerator for the isolation of chlorpyrifos degrading bacteria.
3.2 Characterization of soil samples for chemical properties
The samples were analyzed for their chemical properties like pH, EC and organic
carbon by following standard procedures as given below.
3.2.1 Determination of soil pH
Soil pH determination is important, as it enables to predict the soil fertility,

availability of nutrients and activity of microorganisms in soils. Twenty grams of each
soil sample was taken in 100 ml beakers and 40 ml distilled water was added to each
flask, followed by stirring four times within a period of half an hour and kept for another
half an hour for settlement of soil particles and stirred again followed by settlement.
Stirred soil suspension was measured with a digital pH meter (Piper, 1966).
3.2.2 Determination of Electrical conductivity (EC)
The measurement of soil electrical conductivity is important, as it renders

information about the concentration of soluble salts at a particular temperature in the soil.
Twenty grams of each soil sample was taken in 100 ml beakers, added 40 ml of distilled
water was added to each. It was stirred intermittently for 2-3 minutes and left overnight
for getting a clear supernatant. EC of the supernatant solution was measured using
Conductometric method (Jackson, 1973).
3.2.3 Determination of Organic Carbon (OC)
The soil organic carbon was determined by using a wet oxidation method of
Walkley and Black (1934). Two gram of two mm sieved soil sample was ground in a pestle
and mortar and passed through a 0.2 mm sieve. One gram of this sample was transferred
into 500 ml dry conical flask. To these flasks, ten ml of 0.1 N K2Cr2O7 was added and
mixed well. By adding 20 ml of conc. H2SO4, these flasks were swirled for 2-3 min and
allowed to stand for 30 min on an asbestos sheet. Two hundred ml of distilled water was
poured to dilute the suspension. Ten ml of 85 per cent H3PO4 and one ml
(10 drops) of diphenylamine indicator was added which gave violet color to the suspension.
Contents of the flask were titrated against 0.5 N ferrous ammonium sulfate solutions taken
in burette until the color changed from violet through dark blue to bright green. A blank
reading was also taken without soil. The per cent organic carbon in the soil was calculated
by using the formula.
Organic carbon (%) = N of FAS × (BTV-STV) × 0.003 × 100 × 1N K2Cr2O7 × 1.33

Weight of soil sample (g)
Where,
STV = Blank Titrated Value
BTV = Sample Titrated Value
SAS = Ferrous Ammonium Sulphate

3.3 Isolation and purification of chlorpyrifos degrading bacteria from the
rhizosphere of rice
The chlorpyrifos degrading bacteria were isolated from the collected soil samples
using an enrichment culture technique. The soil samples were serially diluted and plated
on minimal salt agar plates amended with 200 ppm of chlorpyrifos as the sole source of
carbon for the isolation of chlorpyrifos degrading bacteria (Vijayalakshmi and Usha,
2012). The plates were incubated at room temperature (30 ± 1 °C) for 7 days. The colonies
exhibiting proper growth on the plates were selected and purified by four-way streak plate
method. The single isolated colonies of isolates from four-way streak plate were
transferred to nutrient agar slants for further experiments.
3.4 In vitro screening for the growth of chlorpyrifos degrading bacteria in MSM
broth by enrichment culture technique
The isolates obtained from individual colonies were inoculated into flasks
containing 100 ml MSM broth supplemented with 300 ppm concentration of chlorpyrifos
and were incubated on a rotary shaker at 150 rpm for seven days at 30 °C. After seven days
of shaking, aliquot of broth was taken from the flasks and the turbidity was tested using
spectrophotometer at OD600. Simultaneously, 10 µl of the broth was plated on nutrient agar
(without chlorpyrifos) to confirm the growth of the bacteria. Similarly, the isolates were
further screened using enrichment culture technique by supplementing higher
concentrations chlorpyrifos in the media (400 ppm and 600 ppm) to obtain efficient strains.
3.5 Estimation of percent chlorpyrifos degradation by efficient isolates under in

vitro conditions
3.5.1 Inoculum preparation
Each isolate was grown in nutrient broth containing 200 ppm of chlorpyrifos.
After 2 days of incubation, the cultures were centrifuged at 4600 rpm for 5 min, washed
and then diluted with distilled H2O. Colony forming units (CFU) ml−1 of the suspensions
were determined by the dilution plate counting method. For pesticide biodegradation
studies, a cell concentration corresponding to 1 × 107 CFU ml−1 were used so as to
maintain uniformity in cell number.
3.5.2 In vitro estimation of percent chlorpyrifos degradation by efficient isolates
The flasks (250 mL) containing mineral salt medium (100 ml) supplemented with
200 ppm concentration of chlorpyrifos were inoculated with bacterial cell suspension in
triplicates. The flasks were incubated at 30 °C with shaking at 150 rpm and an
uninoculated flask was used as a control. An aliquot of the culture was withdrawn at
regular intervals of 2 days each for 10 continuous days and centrifuged at 10000 rpm for
5 minutes. The pellet was discarded and the supernatant was retained. Three ml of cell-
free extract was tested for chlorpyrifos degradation analysis by observing under UV
spectrophotometer at OD300. Per cent degradation of the compound was determined by
using the formula,
Percent degradation = Ab - Aa × 100

Ab
Where,
Ab = Initial absorbance of compound
Aa = Final absorbance of compound
3.6.1 Morphological characterization of the efficient chlorpyrifos degrading

bacteria
3.6.1.1 Colony characters of the bacterial isolates
All the selected isolates were examined for the colony morphology such as color
(white, cream, yellow, creamish yellow, brown, light brown and pink), size (pinpointed,
small, large and very large), form (circular, irregular, filamentous and rhizoid) and surface
(smooth, rough, glistening and dull).
3.6.1.2 Microscopic characters of the bacterial isolates
The cell shape, motility and gram reactions were carried out as per the standard
procedures given by Barthalomew and Mittewer (1950).
3.6.1.2.1 Shape
The cell shape was observed by simple staining. A smear of each isolate was made,
air dried and stained with crystal violet for 30 sec. The stained smear was washed, air dried
and observed under oil immersion compound microscope.
3.6.1.2.2 Motility
The motility of cells was observed directly under a microscope using a slide with
concave circular space, where a drop of bacterial culture was added and covered with a
coverslip. The slide was inverted carefully and motility was observed.
3.6.1.2.3 Gram staining
The chlorpyrifos degrading bacterial isolates were smeared on a clean glass slide
and treated with crystal violet solution for 1 min, the smear was gently rinsed off using
distilled water and iodine solution was applied for 1 min. This, in turn, was drained off and
the smear was decolorized using ethyl alcohol (95 %) for 10 sec. Safranin was used as a
counterstain for 45 sec. Then slide was gently rinsed off with water and blotted off. The
slide was observed under a microscope for determining the gram reaction of isolates.
3.6.2 Biochemical characterization of the efficient chlorpyrifos degrading bacteria
The biochemical characterization of the isolates was carried out as per the
procedures outlined by Cappuccino and Sherman (1992). The tests are detailed below.
3.6.2.1 Starch hydrolysis
The ability of the isolates to hydrolyze starch was examined in the petri plates
containing starch agar. Inoculated with test cultures and incubated at 30 °C for three days.
After incubation, the plates were flooded with Lugol’s iodine solution and allowed to
stand for 15-20 minutes. The clear zone around the colony was considered as positive for
the test (Eckford, 1927).
3.6.2.2 Catalase test
The nutrient agar slants were inoculated with test organisms and incubated at
30 °C for 24 hours. After incubation, the tubes were flooded with one ml of three percent
hydrogen peroxide and observed for production of gas bubbles. The occurrence of gas
bubbles was scored positive for catalase activity (Cowan and Steel, 1970).
3.6.2.3 Urease test
The bacterial isolates were tested for urease activity by inoculating the cultures to
five ml of pre-sterilized urea broth containing phenol red as a pH indicator. The tubes
were incubated for 24 to 48 hours at 30 °C. The formation of dark pink color was taken
as positive for urease activity (James and Sherman, 1992).
3.6.2.4 Methyl red test
The test tubes containing pre-sterilized MR-VP broth were inoculated with the
test cultures and they were incubated at 28 ± 2 °C for 48 hours. After incubation, five
drops of methyl red indicator were added to each tube and gently shaken. The production
of red color was taken as positive for the test and production of yellow color was taken
as negative for the test (Seeley and Vandemark, 1981).
3.6.2.5 Voger-Proskauer test
To the pre-sterilized tubes containing MR-VP broth, the test cultures were
inoculated. The tubes were incubated for 48 hours at 37 °C. After incubation ten drops of
Barritt’s reagent A was added and gently shaken followed by addition of ten drops of
Baritt’s reagent B. The development of rose color in the broth was taken as positive for
the test (Seeley and Vandemark, 1981).
3.6.2.6 Citrate Utilization
The isolates were streaked on to sterilized Simmon’s citrate agar slants and
incubated at 28 ± 2 °C for 24 h. Change in color from green to blue indicates the positive
reaction for citrate utilization (Simmons, 1926).
3.6.2.7 Denitrification test
The nitrate broth tubes with inverted Durham’s tube inside were inoculated with
the overnight grown culture of the test organism and that was incubated for one week at
25 °C. After one week of incubation, the inverted Durham’s tube was observed for the
accumulation of gas (James and Sherman, 1992).
3.6.2.8 Indole production
To the pre-Sterilized Sulfide Indole Motility (SIM) agar tubes, the test cultures
were inoculated and the tubes were incubated at 28 ± 2 °C for 48 hours. After incubation,
each tube was added with 10 drops of Kovac’s reagent. The production of red color was
taken as positive for the indole production (Cowan and Steel, 1970).
3.6.2.9 Hydrogen Sulphide production
The bacterial isolates were inoculated into test tubes containing 5 ml of sterile
SIM agar medium and were incubated at room temperature 28 °C. The formation of a
black ring in the medium was taken as positive for H2S production (Cowan and Steel,
1970).
3.6.2.10 Casein hydrolysis
The plates containing pre-sterilized skim milk agar were streaked with test
cultures and incubated at 30 °C for one week. The clear zone around the colony against
a creamy white background after incubation was taken as positive for casein hydrolysis
(Seeley and Vandemark, 1981).
3.6.2.11 Acid and gas production
The isolates will be tested for acid and gas production by inoculating five ml of
pre-sterilized glucose broth medium in test tubes containing Durham’s tube and bromo-
cresol purple (15 ml L-1 of 0.04 % solution) as pH indicator. The tubes were incubated
for seven days at 30 °C. The accumulation of gas in the Durham’s tube was taken as
positive for gas production and the change in color of the medium from purple to yellow
were scored as positive for acid production (Seeley and Vandemark, 1981).
3.6.2.12 Gelatin liquefaction
To the pre-sterilized nutrient gelation deep tubes, the test cultures were inoculated
and tubes were incubated at 28 ± 2 °C for 24 hours. Following this, the tubes were kept
in a refrigerator at 4 °C for 30 minutes. The tubes with cultures that remained liquefied
were taken as positive and those that solidified on refrigeration were taken as negative
for the test (James and Sherman, 1992).
3.7 In vitro screening of the efficient chlorpyrifos degrading bacterial isolates for
their beneficial attributes
The efficient chlorpyrifos degrading bacterial isolates were further used in

elucidating the mechanisms of growth promotion such as the production of IAA,
siderophore production and phosphate solubilization as given below.
3.7.1 Indole Acetic Acid (IAA) production
The IAA production potential of chlorpyrifos degrading bacterial isolates was tested
in nutrient broth supplemented with 0.1 % concentration of tryptophan at 28 °C. After 3
days of incubation, the culture broth was centrifuged at 5,000 rpm for 5 min. The cell-free
supernatant extract was taken and the pellets were discarded. The concentration of IAA in
the culture broth was determined by a spectrophotometric method using Salkowaski’s
reagent as given below.
One ml of the supernatant was mixed with 1 ml of Salkowaski’s reagent (2 ml of

0.5 M FeCl3 + 98 ml 35 percent HClO4) and the intensity of red color developed within 30
min was checked at 530 nm using a spectrophotometer. The concentration was determined
by using a standard curve prepared from a standard solution of indole acetic acid (Gordon
and Weber, 1951).
The siderophore assay was carried out based on the CAS shuttle assay. Isolates were
grown in 100 ml sterilized nutrient broth for two days. The culture broth was centrifuged
at 10,000 rpm for 10 min. After centrifugation, the cell-free supernatant was collected and
the same was subjected to siderophore assay (Payne, 1994).
The culture extract (0.5 ml) was mixed with 0.5 ml of CAS reagent. The color
obtained was measured using a spectrophotometer at 630 nm after 20 min of incubation.
The blank was prepared using uninoculated broth medium. The Siderophore content in the
aliquot was calculated by using the following formula,
Siderophore units (%) = As ₋ Ar × 100

As
Where,
As = Absorbance of sample (culture extract + CAS reagent)
Ar = Absorbance of the reference (CAS reagent)
3.7.3 Phosphate solubilization
The pre-sterilized Pikovskaya’s agar medium was poured as a thin layer on to the
sterilized Petri plates and incubated for 24 h. After incubation, the Pikovskaya’s plates were
spot inoculated with isolates and incubated at 28 ± 1 °C for 4 days. Formation of a clear
zone around the colony was considered as a positive result for phosphate solubilization
(Pikovskaya, 1948).
PSE (Phosphate Solubilization Efficiency) = Z × 100

C
Where,
Z = Clearance zone including bacterial growth
C = Colony diameter
3.8 Evaluation of efficient chlorpyrifos degrading bacteria on growth of paddy

under pot culture
The pot experiment to evaluate the effect of chlorpyrifos degrading bacteria was
carried out in the glasshouse condition at College of Agriculture, Raichur.
Table 1: Treatment details of the pot culture experiment
Sl. No. Particulars Details
1 Design Completely Randomized Design
2 Replications Three
T1-Control
T2- CP
T3- CP + CDB-6
T4- CP + CDB-11
3 Treatment T5- CP + CDB-18
T6- CP + CDB-6 + CDB-11
T7- CP + CDB-11 + CDB-18
T8- CP + CDB-6 + CDB-18
T9- CP + CDB-6 + CDB-11+ CDB-18
4 Crop Paddy
5 Variety BPT-5204
6 Soil type Black vertisols
Note:
CP- Chlorpyrifos; CDB – Chlorpyrifos degrading bacteria
3.8.1 Preparation of inoculants
3.8.1.1 Preparation of mother culture
The efficient inoculants were inoculated into 100 ml of pre-sterilized nutrient

broth and incubated on a shaker at 120 rpm for 3 days. Turbidity develops after two days
and it was used for the preparation of broth culture.
3.8.1.2 Preparation of broth culture
Five percent of the total volume from mother culture was transferred to the pre-
sterilized broth and it was incubated at room temperature for 4 days for development of
inoculum up to 1 × 108 CFU ml-1 in the broth.
3.8.1.3 Mixing with the carrier material
The broth culture was mixed with pre-sterilized carrier material in the ratio of
1:2.5 and was dried under the shade to reduce the moisture to 30-40 % and cured for
about 24 h.
3.8.1.4 Seedling root dipping
The inoculant (200 g) was mixed with 5 liters of water and stirred thoroughly.
The root portion of seedlings was dipped for 5 minutes and transplanted to pots after
5 minutes.
3.8.2 Planting pattern
The pots were filled with sterilized paddy field soil and supplemented with
chlorpyrifos at the conc. of 500 ppm. The uninoculated soils were used as controls. The
seedlings treated with efficient isolates were transplanted in pots according to the
treatments (Table 1). The pots arranged as completely randomized block design with three
replicates. Irrigation, manuring, weeding was followed as per standard method.
3.8.3 Plant growth parameters
The efficient chlorpyrifos degrading bacteria selected from in vitro studies were
treated with the paddy seedlings and transplanted in pots, observations on growth
parameters were recorded at periodical intervals.
3.8.3.1 Plant height (cm)
The plant height was measured from the base of the plant to tip of the topmost leaf
at early stages and from the base of the plant up to the tip of the main panicle at maturity
and expressed in centimeters.
3.8.3.2 Number of tillers hill-1
The total tillers for each hill in all the replications were counted on 30, 60 and 90
days after transplantation (DAT). From this data, the average number of tillers hill-1 was
calculated.
3.8.3.3 Number of leaves hill-1
The total number of leaves hill-1 was counted from individual potted plants on 30,
60 and 90 DAT and mean value was obtained for each treatment.
3.8.3.4 Dry matter yield pot-1 (g)
The plants from each treatment were collected on 30, 60 DAT and at the time of
harvest. The plant samples with shoots, roots and leaves were washed and then air dried in
shade for 24 to 48 hours and then oven dried at 60 °C until constant weight is obtained. The
total dry matter production per plant was obtained with the summation of the dry weight of
all plant parts and expressed in terms of g plant-1.
3.8.4 Determination of microbial population in the soil
The soil samples were weighed to one gram and then serially diluted to 10-5 to 10-6
in 9 ml sterile water blanks. 0.1 ml of suspensions from final dilutions were inoculated on
to nutrient agar plates and incubated at 37 °C for 24 hours in a BOD incubator for the
enumeration of bacteria.
Similarly, 0.1 ml of suspension from 10-3 dilution was plated on Martin Rose Bengal
Agar for isolation of fungi and 0.1 ml of suspension from 10-4 was plated on Kusters Agar
medium for determination of actinomycetes population. The plates were incubated at 37 °C
for 4 days in BOD. The observations were taken at 30th, 60th and 90th DAT and represented
as CFU per gram of soil.
3.8.5 Determination of enzymatic activity in the soil
The soil samples were analyzed for the enzymatic activities viz., dehydrogenase
and phosphatase at 30th, 60th and 90th DAT.
3.8.5.1 Dehydrogenase
2-3-5-Triphenyl Tetrazolium Chloride (TTC) reduction technique was used for the
estimation of dehydrogenase activity in soil. For this, one gram of fresh soil was taken in a
test tube and then mixed with 0.1 g of calcium carbonate (CaCO3) and 1 ml of 1 % TTC
solution. The mixture was then shaken and plugged with a rubber stopper and incubated at
30 °C for 24 hours in an incubator. Three replicates were maintained in each case.
The resulting slurry was transferred on Whatman filter paper No.1 and extracted
with successive aliquots of concentrated methanol. The volume of the filtrate was made to
50 ml by adding methanol. The optical density of the filtrate was read at 485 nm using a
spectrophotometer, using methanol extract as a blank. The activity was represented in terms
of concentration of Formazan, which was calculated by a standard curve of triphenyl
formazan in methanol. Dehydrogenase activity per gram of dry soil was expressed in terms
of microgram formazan per gram of dry soil per 24 hours
(Casida, 1977).
3.8.5.2 Phosphatase
Air-dried soil was weighed to 0.1 g and placed in a 50 ml conical flask. Then 4 ml
of modified universal buffer (pH 6.5), 0.25 ml of toluene and 1 ml of 0.115 M
p-nitrophenyl phosphate (PNP) solutions were added to the flask. The flask was swirled for
a few seconds and then incubated at 37 °C for one hour in an incubator. After incubation,
1 ml of 0.5 M calcium chloride and 4 ml of 0.5 M sodium hydroxide were added to the
mixture. The soil suspension was filtered through Whatman filter paper no. 1. The optical
density of the filtrate was measured at 430 nm using a spectrophotometer. Blank was
maintained similarly without soil. The phosphatase activity in terms of concentration of p-
nitrophenyl in each sample was calculated by a standard curve of
p-nitrophenol in water and was expressed as moles of p-nitrophenol released per gram of
dry soil per hour.
3.8.6 Analysis of chlorpyrifos degradation in soil by the efficient bacterial isolates
using Gas Chromatography-Mass Spectroscopy (GC-MS/MS)
The analysis of chlorpyrifos in the soil samples was carried out using GC-MS/MS
(Make – Shimadzu TQ-8030) with nonpolar Rxi-5Sil MS Column. Helium was used as the
carrier gas and the temperature programming was set as mentioned in the analytical
conditions given below. One microliter of each sample was injected in splitless mode. Mass
spectra were recorded over 50-500 amu range with electron impact ionization energy 70
eV. The total running time for a sample was 40.60 min. Each step involved in the analysis
of chlorpyrifos in soil sample such as standard preparation, sample preparation, extraction,
clean up, evaporation and injection are explained below.
3.8.6.1 Standard preparation
Physical and chemical properties of chlorpyrifos
Common name : Chlorpyrifos

Chemical name :0,0-diethyl0-(3,5,6-trichloro-2 pyridinyl)
- phosphorothioate
Molecular Formula : C9H11Cl3NO3PS (Fig. 1)
Molecular Weight : 350.575 g mol-1
Appearance : Colourless crystals
Odor : mercaptan like
Density : 1.398 g/cm3 943.5 °C)
Melting point : 43 °C
Boiling point : 160 °C
Vapor pressure : 1.87 × 10-5 mmHg at 25 °C
Octanol-Water Partition Coefficient (Kow) : 4.70
Solubility (water) : 0.0014 g/L (1.4 mg/L) at 25 °C
Soil Sorption Coefficient (Koc) : 360 to 31,000
Stability : Stable at recommended storage conditions
3.8.6.1.1 Stock preparation
A stock concentration of 1 mg ml-1 (99 %) was prepared by accurately weighing

10.12 mg of chlorpyrifos certified reference material (CRM) standard into a clean 10 ml
calibrated standard volumetric flask containing 3 ml of ethyl acetate, it was dissolved well
and the final volume of the solution was made up to 10 ml with the solvent. The prepared
chlorpyrifos standard solution was stored in a deep freezer at -20 °C. The final
concentration of the primary stock solution was calculated by using the following formula,
Concentration = Wt. of CRM (mg) × Purity of CRM (%) × 1000 µg ml-1

Final volume 100
3.8.6.1.2 Working standard preparation
An intermediate standard stock concentration of 10 ppm was prepared from the

stock solution by transferring 0.1 ml of stock solution to 9.9 ml of ethyl acetate in a
10 mL standard volumetric flask using the following formula,
N1V1 = N2V2
Where, N1 = Available concentration
V1 = Volume to be taken from the available stock
N2 = Required concentration
V2 = Required volume
From the intermediate standard, a working standard concentration of 100 ppb was
prepared by transferring 0.1 ml from 10 ppm solution to 9.9 ml of ethyl acetate and the
resulted 100 ppb was further used for analysis to calculate the unknown sample
concentration. A calibration curve of chlorpyrifos standard was plotted with the
concentrations ranges from 100 ppb.
3.8.6.2 Sample preparation
The extraction of chlorpyrifos in soil was followed by a soil pesticide-specific

extraction procedure called QuEChERS method of extraction used in the PRFQAL.
3.8.6.2.1 Sample extraction
The soil sample was sieved and weighed to 20 g in 50 ml centrifuge tubes, 12 ml of

distilled water was added and incubated for 30 minutes. Then 20 ml of acetonitrile was
added and kept on a shaker (Horizontal shaker) at 250 rpm for 4 hours. Tubes were
centrifuged at 5000 rpm for 3 minutes at 10 °C.
3.8.6.2.2 Clean-up
The supernatant was transferred to a 50 ml centrifuge tube containing 6 g

Magnesium sulfate, 1.5 g Sodium chloride, 1.5 g Trisodium citrate dehydrate and 750 mg
disodium hydrogen citrate sesquihydrate. Tubes were shaken vigorously for 1 min then the
content was centrifuged at 5000 rpm for 5 minutes at 10 °C. After centrifugation, 6 ml
supernatant was transferred to a 15 ml centrifuge tube containing 150 mg of primary
secondary amine (PSA) and 900 mg of Magnesium sulfate (MgSO4). The mixture was
vortexed for 30 seconds and centrifuged for 5 minutes at 12000 rpm (10 °C).
3.8.6.2.3 Evaporation
The supernatant (2 ml) was transferred to a tube containing 200 µl of 10 %

diethylene glycol in methanol and evaporated the content using nitrogen concentrator at
35-40 °C temperature. The residues were reconstituted with 1.5 ml solvent mixture (ethyl
acetate). The mixture was sonicated for 1 min to dissolve the residue. The content (2 ml)
was filtered through a 0.22 µ PTFE membrane filter for GCMS/MS analysis.
3.8.6.2.4 Injection
The filtrate (1 µL) was injected to GC-MS/MS using the following instrument
parameters.
3.8.6.2.5 Analytical condition
Instrument : Shimadzu GCMS-TQ8030 System coupled

with GC-2010 plus
Column : Rxi-5Sil MS (30 m, 0.25 mm, 0.25 µm)
Carrier gas : Helium
CID gas : Argon
Temp. Program : 60 °C/2 min, (25 °C) 150 °C/0 min, (3°C) 200°C/0 min, (8 °C)
280 °C/5 min, and (15 °C) 300 °C/2 min
Injection temperature : 300 °C Ion source : 220 °C
Column temperature : 60 °C Interface : 280 °C
Interface : DI Injection mode : Split-less
Detector voltage : 0.45 kV Mode : MRM
Injection
Analytical run time : 40.60 min : 1 µL
Volume
The residual concentration and degradation (%) of chlorpyrifos after 30 days of
treatment was calculated by using the formula,
Sample peak area × Conc. of std. injected (ppm) × Std injected (µl) × Final volume
Residues = of the sample (ml)
(mg kg-1) Std peak area × Weight of sample (g) × Sample injected (µl)
Sample weight (20 g) × Aliquot taken (ml)

Weight of the sample =
(g) Volume of extractant (ml)
Degradation (%) = (Initial Conc. – Residual conc. after 30 days) × 100

Initial conc.
Experimental Results
IV. EXPERIMENTAL RESULTS
In this present investigation, chlorpyrifos degrading bacterial isolates were isolated

from rhizosphere of rice grown in TBP command area. The efficient isolates were selected
based on in vitro efficiency of chlorpyrifos degradation at higher concentration as well as
their beneficial traits. Further they were characterized and evaluated for their effect on
growth attributes of paddy in pot culture conditions. The data obtained from in vitro and in
vivo experiments are presented below.
A total of forty rhizospheric samples from 14 locations (Fig. 2) were collected from
top soil of rhizosphere of rice ranging in depth from 10-15 cm in sterilized polythene bags.
Samples from each site were collected and stored in deep freezer at 4 °C in laboratory. All
forty samples collected were black soils (Table 2). They were further subjected to physico-
chemical analysis.
4.2 Characterization of soil samples for chemical properties
All the rhizospheric soil samples collected from 14 different locations of TBP
command area were analysed for their chemical properties like pH, EC and organic carbon
by following standard procedures. In the present study physico-chemical analysis of soil
samples revealed a range of pH 6.95 to 8.88, electrical conductivity (EC) varied from 0.12
to 0.92 dSm-1 while percent organic carbon ranged from 3.18 % to 6.75 % in forty soil
samples. Results of this analysis are mentioned in Table 3.
4.3 Isolation and purification of chlorpyrifos degrading bacteria from the

rhizosphere of rice
Twenty-five chlorpyrifos degrading bacteria were isolated from different

rhizosphere soils of rice grown in TBP command area. After 7 days of incubation at 30⁰C,
observed a profused growth of the bacterial colonies on MSM agar plates supplemented
with 200 ppm of chlorpyrifos as sole source of carbon. The bacterial isolates which showed
prolific growth and distinct colony morphology were picked up and purified by repeated
streaking on nutrient agar plates. The purified isolates thus obtained were
Fig. 1. Structure of chlorpyrifos
Fig. 2. Google map showing the areas of soil samples collected for the isolation of
chlorpyrifos degrading bacteria
Table 2: Details of location, soil type and source of soil samples used for isolation of
chlorpyrifos degrading bacteria
Sl. No. Location District Soil type Source Sample code
1 Maliyabad Raichur Black soil Rice rhizosphere MLBD- 1
2 Maliyabad Raichur Black soil Rice rhizosphere MLBD-2
3 Yeragera Raichur Black soil Rice rhizosphere YRGR- 1
4 Yeragera Raichur Black soil Rice rhizosphere YRGR- 2
5 Mantralaya Raichur Black soil Rice rhizosphere MNTY- 1
8 Kalmala Raichur Black soil Rice rhizosphere KLML- 1
11 Manavi Raichur Black soil Rice rhizosphere MNVI-1
14 Siravar Raichur Black soil Rice rhizosphere SRVR- 1
17 Neer manavi Raichur Black soil Rice rhizosphere NRMV- 1
20 Kallur Raichur Black soil Rice rhizosphere KLUR- 1

23 Kapagal Raichur Black soil Rice rhizosphere KPGL- 1
26 Kavital Raichur Black soil Rice rhizosphere KVTL- 1
27 Kavital Raichur Black soil Rice rhizosphere KVTL- 2
28 Tuntapur Raichur Black soil Rice rhizosphere TNPR- 1
31 Sindhanur Raichur Black soil Rice rhizosphere SNDR- 1
35 Gangavati Koppal Black soil Rice rhizosphere GNVT- 1
39 Siddapur Koppal Black soil Rice rhizosphere SDPR- 1
40 Siddapur Koppal Black soil Rice rhizosphere SDPR- 2

Table 3: Chemical properties of paddy rhizospheric soil samples collected from
Tungabhadra command area
Sl. No. Sample code pH EC (dS m-1) OC (g kg-1)
1 MLBD-1 7.85 0.57 5.73
2 MLBD-2 7.24 0.46 5.93
3 YRGR-1 7.60 0.38 5.70
4 YRGR-2 6.95 0.12 4.63
5 MNTY- 1 6.98 0.37 4.97
6 MNTY-2 7.30 0.51 6.36
7 MNTY-3 7.47 0.56 4.45
8 KLML-1 7.60 0.61 6.75
9 KLML-2 7.56 0.29 4.30
10 KLML-3 8.3 0.82 3.96
11 MNVI-1 7.82 0.55 3.92
12 MNVI-2 7.70 0.34 4.50
13 MNVI-3 8.00 0.43 4.75
14 SRVR-1 7.34 0.51 3.97
15 SRVR-2 7.70 0.56 4.22
16 SRVR-3 7.78 0.60 4.75
17 NRMV-1 7.73 0.55 3.65
18 NRMV-2 7.64 0.54 3.35
19 NRMV-3 7.65 0.53 3.49
20 KLUR-1 7.57 0.60 3.77

21 KLUR-2 7.75 0.63 4.91
22 KLUR-3 8.60 0.77 3.75
23 KPGL-1 7.91 0.41 3.56
24 KPGL-2 7.55 0.57 4.25
25 KPGL-3 8.10 0.61 5.30
26 KVTL-1 8.10 0.68 5.16
27 KVTL-2 8.04 0.67 3.95
28 TNPR-1 7.67 0.59 3.65
29 TNPR-2 7.70 0.54 3.50
30 TNPR-3 7.47 0.58 3.18
31 SNDR-1 8.02 0.74 4.41
32 SNDR-2 7.85 0.69 3.97
33 SNDR-3 7.96 0.71 4.00
34 SNDR-4 7.82 0.65 4.95
35 GNVT-1 8.52 0.89 3.54
36 GNVT-2 8.12 0.84 3.69
37 GNVT-3 8.21 0.85 5.10
38 GNVT-4 8.88 0.92 3.44
39 SDPR-1 8.23 0.75 4.85
40 SDPR-2 7.98 0.71 4.31

Plate 1: Isolated colonies of chlorpyrifos degrading bacteria on MSM agar
Plate 2: Purification of the chlorpyrifos degrading bacterial isolates

Plate 3: Pure culture slants of the isolated 25 chlorpyrifos degrading bacteria
maintained on agar slants prepared with nutrient medium (Plate 1-3). These 25 isolates
were from 14 different locations.
4.4 In vitro screening for growth of chlorpyrifos degrading bacterial isolates in

minimal salt medium by enrichment culture method
Out of 100 isolates that could grow on MSM agar plates supplemented with 200
ppm chlorpyrifos, 25 best isolates were selected based on their profused growth noticed on
the medium and were further screened for their ability to grow in MSM media
supplemented with higher concentrations of chlorpyrifos.
When the aliquot of the culture broth was examined for the development of turbidity
using spectrophotometer at OD600, out of 25 isolates, eleven isolates were able to increase
the turbidity of the media containing 300 ppm of CP, showing positive results for growth
in the media (Table 4).
The eleven isolates which could grow at 300 ppm of chlorpyrifos (CP) were
subjected to further screening at the concentration of 400 ppm of CP. When the isolates
were inoculated in MSM media supplemented with 400 ppm of CP and incubated for a
week, nine isolates namely CDB-1, CDB-3, CDB-6, CDB-11, CDB-12, CDB-14, CDB-16,
CDB-18 and CDB-19 showed comparatively higher turbidity than the control and also
confirmed growth on NA plates (Table 5).
The nine isolates which responded positively for the growth test at 400 ppm CP
supplement were tested at the higher concentration of CP (600 ppm). Among them, seven
isolates namely, CDB-1, CDB-6, CDB-11, CDB-14, CDB-16, CDB-18 and CDB-19
showed comparatively higher turbidity than control (Table 6) and were selected for the
percent biodegradation studies.
4.5 Estimation of percent chlorpyrifos degradation by efficient isolates under in

vitro conditions
The efficient isolates with cell concentration corresponding to 1 × 107 CFU ml−1
were analysed for the biodegradation ability using mineral salt medium (100 ml)
Table 4: In vitro screening for growth of chlorpyrifos degrading bacterial isolates in
minimal salt medium supplemented with 300 ppm CP concentration
Growth of isolates at 300 ppm chlorpyrifos

Sl. No. Isolate
OD (600) Growth on NA plates
1 CDB-1 1.401 Positive
4 CDB-4 0.461 Negative
26 Control 0.450 Negative
Table 5: Invitro screening for growth of chlorpyrifos degrading bacterial isolates in
Growth of isolates at 400 ppm

chlorpyrifos
Sl. No. Isolate
Growth on nutrient
OD (600)
agar plates

Table 6: Invitro screening for growth of chlorpyrifos degrading bacterial isolates in
Growth of isolates at 600 ppm

chlorpyrifos
Sl. No. Isolate
Growth on nutrient
OD (600)
agar plates

supplemented with 200 ppm concentration of chlorpyrifos using a spectrophotometer
(Table 7).
The total degradation of chlorpyrifos by all the efficient isolates at the end of ten
days ranges between 50.5 % and 72.4 %. Highest degradation was showed by CDB-11 with
72.4 % followed by CDB-18 and CDB-6 which showed chlorpyrifos degradation up to 70.2
% and 68.5 %, respectively and the lowest was recorded by CDB-1 (50.5 %). Concentration
of chlorpyrifos in the control showed 2 % degradation at the end of
10 days span as shown in Table: 7.
The inoculation of CDB-11 resulted in decrease of the chlorpyrifos (CP)

concentration from 200 to 177.4 ppm within 2 days. After 4th, 6th and 8th day, CP
concentration was brought down to 137.8, 107.4 and 73.8 ppm, respectively. By the end of
10th day, chlorpyrifos was reduced to 55.2 ppm.
The isolate CDB-18 brought down the chlorpyrifos concentration from 200 to 191.4
ppm within 2 days. After 4th, 6th and 8th day, the CP concentration was reduced to 169.6,
131.4 and 76.2 ppm, respectively. On the 10th day, chlorpyrifos was declined to
59.6 ppm.
The inoculation of CDB-6 reduced the chlorpyrifos concentration from 200 to 183.2
ppm within 2 days. There was a drop in the concentration to 142.2, 144 and 82.6 ppm
during 4th, 6th and 8th day, respectively. By the end of 10th day, chlorpyrifos was decreased
to 63 ppm (Table 7).
4.6 Characterization of efficient chlorpyrifos degrading bacterial isolates
4.6.1 Morphological and cultural characterization of the efficient chlorpyrifos

degrading bacterial isolates
The cell morphology, colony morphology and gram reaction were studied for
eleven chlorpyrifos degrading isolates. Among them, three isolates were found to be gram
positive (CDB-3, CDB-12 and CDB-17) and remaining eight isolates (CDB-1, CDB-2,
Table 7: Degradation percentage of chlorpyrifos at 200 ppm concentration in MSM broth inoculated with efficient bacterial isolates at
different time intervals
CDB-1 CDB-6 CDB-11 CDB-14 CDB-16 CDB-18 CDB-19

Time of
interval Conc.
Conc. Conc. Conc. Conc. Conc. Conc.
(%) (%) (%) (%) (ppm) (%) (%) (%)
(ppm) (ppm) (ppm) (ppm) (ppm) (ppm)
Initial 200 Nil 200 Nil 200 Nil 200 Nil 200 Nil 200 Nil 200 Nil
2nd day 185.8 7.1 183.2 8.4 177.4 11.3 183.4 8.3 193.4 3.3 191.4 4.3 185.2 7.4
4th day 171.6 14.2 142.2 28.9 137.8 31.1 148 26.0 167.4 16.3 169.6 15.2 150 25.0
6th day 136.4 31.8 114 43.0 107.4 46.3 124.4 37.8 142 29.0 131.4 34.3 119.6 40.2
8th day 118 41.0 82.6 58.7 73.8 63.1 98.8 50.6 114 43.0 76.2 61.9 94.8 52.6
10th day 99 50.5 63 68.5 55.2 72.4 82.6 58.7 88.8 55.6 59.6 70.2 77.4 61.3
Table 8: Morphological characteristics of the efficient chlorpyrifos degrading
bacterial isolates isolated from rhizosphere soil of rice
Morphological characterization
Sl.
Isolate Motility
No. Gram
Colony character
reaction
Creamy White, Circular, Medium, Smooth,
1 CDB-1 -ve, rod Motile
Flat
2 CDB-2 Yellow, Irregular, Large, Smooth, Flat -ve, rod Motile
Creamy, Circular, Medium, Smooth,

3 CDB-3 +ve, rod Motile
Convex
4 CDB-6 White, Circular, Small, Smooth, Raised -ve, rod Motile
Non
5 CDB-11 Yellow, Irregular, Large, Smooth, Convex -ve, rod
motile
6 CDB-12 White, Irregular, Large, Smooth, Convex +ve, rod Motile
7 CDB-14 Light Yellow, Circular, Small, Rough, Flat -ve, rod Motile
8 CDB-16 White, Circular, Small, Smooth, Raised -ve, rod Motile
Creamy White, Circular, Large, Smooth, Non

9 CDB-17 +ve, rod
Flat motile
10 CDB-18 Creamy, Circular, Large, Smooth, Convex -ve, rod Motile
White, Irregular, Small, Rough, Spreading,

11 CDB-19 -ve, rod Motile
Raised
CDB-6, CDB-11, CDB-14, CDB-16, CDB-18 and CDB-19) were gram negative. Microscopic
observation revealed that all eleven isolates were found to be rod shaped and motility test
conducted for these isolates revealed that nine isolates were motile and two isolates i.e.
CDB-11 and CDB-17 were found to non-motile. Each isolate was identified according to
specific characters described in Bergey′s Manual of Systematic Bacteriology.
4.6.2 Biochemical characterization of the efficient chlorpyrifos degrading bacterial

isolates
All the 11 chlorpyrifos degrading bacterial isolates were subjected to different

biochemical tests as shown in Table 9.
4.6.2.1 Starch hydrolysis
All the 11 isolates showed positive results for starch hydrolysis test (Plate 4).
4.6.2.2 Catalase test
All the 11 isolates showed positive results for catalase test (Plate 5).
4.6.2.3 Urease activity
None of the 11 isolates showed positive results for urease activity (Table 9).
4.6.2.4 Methyl red test
None of the 11 isolates showed positive results for methyl red test (Table 9).
4.6.2.5 Voges-Proskauer test
Three isolates (CDB-3, CDB12 and CDB-17) tested positive and remaining eight
isolates (CDB-1, CDB-2, CDB-6, CDB-11, CDB-14, CDB-16, CDB-18 and CDB-19) showed
negative results for Voges-Proskauer test (Plate 6).
4.6.2.6 Citrate utilization test
All the 11 isolates showed positive results for Citrate utilization test (Plate 7)).
Table 9: Biochemical characteristics of the efficient chlorpyrifos degrading bacterial isolates isolated from rhizosphere soil of rice
Biochemical characterization
Sl. No. Isolate Tentative genus
1 2 3 4 5 6 7 8 9 10 11 12
1 CDB-1 + + - - - + + - - + + + Pseudomonas sp.
3 CDB-3 + + - - + + + - + + + + Bacillus sp.
4 CDB-6 + + - - - + + - - - + - Pseudomonas sp.
5 CDB-11 + + - - - + + - - - + + Pseudomonas sp.
6 CDB-12 + + - - + + + - + + + + Bacillus sp.
9 CDB-17 + + - - + + + - + + + + Bacillus sp.
11 CDB-19 + + - - - + + - - + + - Pseudomonas sp.
1 - Starch hydrolysis, 2 - Catalase test, 3 - Urease activity, 4 - Methyl red test, 5 – Voges Proskauer test, 6 - Citrate utilization test, 7 - Denitrification test, 8 - Indole test,
9 - H2S production, 10 - Casein hydrolysis test, 11 - Gas production, 12 - Gelatin liquification
Control CDB-6 CDB-11
CDB-18 CDB-19 CDB-16

Plate 4. Hallow zones around the colonies showing positive for starch hydrolysis test
Plate 5: Production of foam indicating catalase activity

Control CDB-1 CDB-3 CDB-6 CDB-11 CDB-12 CDB-14 CDB-16 CDB-17 CDB-18 CDB-19
Plate 6: Blue coloration of the media showing positive for citrate utilization test
Control CDB-1 CDB-6 CDB-11 CDB-14 CDB-18
Plate 7: Yellow colouration in MR-VP broth indicating negative for VP test

4.6.2.7 Denitrification test
All the 11 isolates showed positive results for denitrification test (Table 9).
4.6.2.8 Indole test
All the 11 isolates showed negative results for indole test (Table 9).
4.6.2.9 H2S production
Three isolates (CDB-3, CDB-12 and CDB-17) tested positive and remaining eight
isolates (CDB-1, CDB-2, CDB-6, CDB-11, CDB-14, CDB-16, CDB-18 and CDB-19) showed
negative results (Table 9).
4.6.2.10 Casein hydrolysis test
Four isolates (CDB-6, CDB-11, CDB-14 and CDB-18) showed negative results for
caseinase test, remaining seven (CDB-1, CDB-2, CDB-3, CDB-12 CDB-16, CDB-17 and CDB-
19) isolates showed positive results for casein hydrolysis test (Table 9).
4.6.2.11 Gas production
All the 11 isolates showed positive results for gas production test (Table 9).
4.6.2.12 Gelatin liquification
Two isolates (CDB-6 and CDB-19) showed negative for the test, remaining nine
isolates showed positive results for gelatin liquification test as shown in the Table 9.
4.7 In vitro screening of the efficient chlorpyrifos degrading bacteria for their
plant growth promoting attributes
The production of IAA, siderophore and phosphate solubilization are important

beneficial traits of organisms which play important role in enhancing the plant growth. In
vitro screening of all 11 chlorpyrifos degrading bacteria for their beneficial traits were
conducted and the results of each investigation are presented below.
When the chlorpyrifos degrading bacterial isolates were grown in culture medium
supplemented with tryptophan as precursor, all the isolates produced IAA as detected by
the Salkowaski’s reagent using spectrophotometer at OD530 (Plate 8).
All the isolates produced IAA whose results are depicted in Table 10. It ranged
between 10.59 μg ml-1 and 20.10 μg ml-1. Significantly higher concentration of IAA was
produced by CDB-11 (20.10 μg ml-1) which is followed by CDB-18 (18.51 μg ml-1). There
was no significant difference between CDB-6 and CDB-19 (17.630 and 17.04 μg ml-1,
respectively). Minimum concentration was recorded in CDB-12 (10.59 μg ml-1).
All the chlorpyrifos degrading bacterial isolates produced siderophore and it was
determined quantitatively based on CAS shuttle assay of Payne (1994) (Plate 9).
The values of siderophore units ranged between 51.33 % and 64.33 % as shown in
the Table: 10. The isolate CDB-11 produced significantly higher siderophore of 64.33 %
followed by the CDB-18 (61.33 %). On par results were observed between CDB-1, CDB-
12 and CDB-14 (59.33, 59.33 and 60.33 %, respectively). Lowest siderophore units were
recorded by CDB-3 (51.33 %).
All the eleven bacterial isolates were able to solubilize phosphate when spot
inoculated on to Pikovskaya’s media plates containing tri calcium phosphate. The zone of
solubilization produced by the isolates were in the range of 8.33 mm to 17.66 mm which is
mentioned in the Table 11.
Among eleven bacterial isolates CDB-18 showed the highest solubilization zone of
17.66 mm followed by CDB-11 (16.66 mm) (Plate 10). Least zone of solubilization (8.33
mm) was formed by isolates CDB-3 and CDB-14 with solubilization efficiency of 113.64
%. Highest solubilization efficiency was shown by CDB-11 (135.11 %) followed by CDB-
18 which showed 132.48 % efficiency.
Table 10: IAA and siderophore production by the efficient chlorpyrifos degrading
bacterial isolates
Production of IAA Siderophore production

Sl. No. Isolate
Qualitative Conc. Qualitative Conc.
test (μg ml-1) test (%)
1 CDB-1 + 15.59 + 59.33
2 CDB-2 + 13.30 + 54.33
3 CDB-3 + 12.47 + 51.33
4 CDB-6 + 17.63 + 59.66
5 CDB-11 + 20.10 + 64.33
6 CDB-12 + 10.59 + 59.33
7 CDB-14 + 15.48 + 60.33
8 CDB-16 + 11.40 + 56.66
9 CDB-17 + 14.51 + 54.66
10 CDB-18 + 18.51 + 61.33
11 CDB-19 + 17.04 + 58.33
S.Em± 0.36 0.33
CD at 1 % 1.46 1.328
Note: CDB: Chlorpyrifos degrading bacteria; Values are mean of three replications.
Control CDB-1 CDB-6 CDB-11 CDB-14 CDB-16 CDB-18
Plate 8: Production of IAA by the efficient chlorpyrifos degrading bacteria
Control CDB-1 CDB-3 CDB-6 CDB-11 CDB-14 CDB-16 CDB-18 CDB-19
Plate 9: Production of siderophore by the efficient chlorpyrifos degrading bacteria

Table 11: Phosphate solubilization by efficient chlorpyrifos degrading bacterial
isolates
Phosphate solubilization Solubilization

Sl. No. Isolate efficiency
Solubilization Culture
zone (mm) diameter (mm) (%)
1 CDB-1 11.66 9.33 124.97
2 CDB-2 10.33 8.33 124.00
3 CDB-3 8.33 7.33 113.64
4 CDB-6 14.66 11.66 125.72
5 CDB-11 16.66 12.33 135.11
6 CDB-12 9.66 8.33 115.96
7 CDB-14 8.33 7.33 113.64
8 CDB-16 11.66 10.33 112.87
9 CDB-17 8.66 8.33 103.96
10 CDB-18 17.66 13.33 132.48
11 CDB-19 11.33 9.33 121.43
S.Em± 0.33 0.37
CD at 1% 1.32 1.49
Note: CDB: Chlorpyrifos degrading bacteria; Values are mean of three replications.
Plate 10: Phosphate solubilization by the efficient chlorpyrifos degrading bacteria
Plate 11: Overall view of pot culture experiment

4.8 Evaluation of efficient chlorpyrifos degrading bacteria on growth parameters
of rice under pot culture
The chlorpyrifos degrading bacterial inoculants were prepared as per the standard
procedure. The root portions of the rice seedlings were dipped in the slurry and planted in
the pots containing the chlorpyrifos at 500 ppm. The uninoculated pots were served as
control (Table 1). Over all view of the pot culture experiment is shown in Plate 11
4.8.1 Plant growth parameters
4.8.1.1 Plant height
The effect of chlorpyrifos degrading bacteria on plant height of rice is presented in

Table 12.
Inoculation of chlorpyrifos degrading bacterial inoculants either singly or in

combination enhanced plant height. On the 30th day of transplanting, the data indicated
that there was a significant difference between combined inoculation of three chlorpyrifos
degrading bacterial isolates when compared to treatments with individual inoculations. The
treatment T9 (CP + CDB-6 + CDB-11 + CDB-18) showed maximum plant height of 29.45
cm followed by treatment T7 (CP + CDB-11 + CDB-18) with 27.50 cm. The individual
inoculation in treatment T3 (CP + CDB-6) showed a plant height of 23.43 cm whereas
treatment T4 (CP + CDB-11) and T5 (CP + CDB-18) recorded plant height of 25.20 and
24.13 cm, respectively. However, there was no significant difference between each other.
Dual combination of inoculants in T6 (CP + CDB-6 + CDB-11) and in T8
(CP + CDB-11+ CDB-18) recorded plant height of 27.37 and 26.34 cm, respectively. The
pots with the sole treatment of chlorpyrifos (T2) recorded lower plant height of 18.49 cm
compared to control (24.60 cm).
On the 60th day of transplanting, the combined inoculation in treatment T9 showed

maximum plant height of 55.51 cm followed by T7 which recorded 53.61 cm. Combined
inoculations were found to be superior on 60th day as well. The individual treatments T3,
T4 and T5 showed the plant height of 43.42, 46.07 and 44.17 cm, respectively. Combination
of two inoculants T6 and T8 showed plant height up to 51.12 and 49.51 cm, respectively. The
pots with the sole treatment of chlorpyrifos (T2) recorded the lowest plant height of 35.43
cm compared to control (45.54 cm).
Table 12: Plant height of rice as influenced by the application of efficient chlorpyrifos
degrading bacterial inoculants
Plant height (cm)

Treatment
30 DAT 60 DAT 90 DAT
T1: Control 24.60 45.54 69.49
T2: CP 18.49 35.43 65.46
T3: CP + CDB-6 23.43 43.42 73.10
T4: CP + CDB-11 25.20 46.07 78.31
T5: CP + CDB-18 24.13 44.17 75.35
T6: CP + CDB-6 + CDB-11 27.37 51.12 81.20
T7: CP + CDB-11 + CDB-18 27.50 53.61 83.49
T8: CP + CDB-6 + CDB-18 26.34 49.51 80.17
T9: CP + CDB-6 + CDB-11+ CDB-18 29.45 55.51 86.37
S.Em+ 0.52 0.49 0.54
CD at 1% 2.13 2.01 2.20
Note:
CP: Chlorpyrifos; CDB: Chlorpyrifos degrading bacteria; DAT: Days after transplanting;
Values are mean of three replications.
On the 90th day of transplanting, the individual inoculation of inoculants in
treatments T3, T4 and T5 showed a plant height of 73.10, 78.31 and 75.35 cm, respectively
and found significant to each other. Similarly, Combination of two inoculants T6, T7 and
T8 showed plant height of 81.20, 83.49 and 80.17 cm, respectively. However, combined
inoculation of all three inoculants (T9) was significantly superior over other inoculations
which recorded plant height of 86.37 cm. The pots with the sole treatment of chlorpyrifos
(T2) recorded the lowest plant height of 65.46 cm while control showed 69.49 cm.
4.8.1.2 Number of tillers per hill
The data revealing the influence of chlorpyrifos degrading bacteria on number of

tillers per hill of rice is presented in Table 13.
On the 30th day of transplanting, the data indicated that there was a significant
difference between combined inoculation of three chlorpyrifos degrading bacteria when
compared to treatments with individual inoculation. The combined inoculation in treatment
T9 showed maximum number of tillers hill-1 i.e. 7.3 followed by treatment T7 with 6.6 tillers
hill-1. The individual inoculation in treatment T3 showed of 5.3 tillers hill-1 whereas
treatment T4 and T5 recorded 5.7 and 4.7 tillers hill-1, respectively and these three treatments
were non-significant to each other. Combination of two inoculants T6 and T8 recorded 6.3
and 6.0 tillers hill-1, respectively. The pots with the sole treatment of chlorpyrifos (T2)
recorded lower numbers of tillers hill-1 i.e. 3.7 compared to control (5.0).

maximum number of tillers hill-1 i.e. 9.7 followed by T7 which recorded 8.3 tillers hill-1.
Combined inoculations were superior and significantly different when compared with
individual inoculation. The individual inoculation of inoculants in treatments T3, T4 and T5
showed 6.3, 6.6 and 5.7 tillers hill-1 respectively, which were non-significant to each other.
Combination of two inoculants T6 and T8 showed 7.3 and 7.0 tillers hill-1 respectively. The
pots with the sole treatment of chlorpyrifos (T2) recorded a lower number of tillers hill-1 i.e.
4.3 compared to control (6.7).
On the 90th day, the individual inoculation in treatments T3, T4 and T5 recorded 7.3,
8.7 and 7.6 tillers hill-1, respectively. Whereas, T4 was significant to the other two. Similarly,
Combination of two inoculants T6, T7 and T8 showed 9.6, 10.0 and 9.3 tillers
Table 13: Number of tillers per hill of rice as influenced by the application of efficient
Number of tillers hill-1

Treatment
T1: Control 5.0 6.7 7.7
T2: CP 3.7 4.3 5.0
T3: CP + CDB-6 5.3 6.3 7.3
T4: CP + CDB-11 5.7 6.6 8.7
T5: CP + CDB-18 4.7 5.7 7.6
T6: CP + CDB-6 + CDB-11 6.3 7.3 9.6
T7: CP + CDB-11+ CDB-18 6.6 8.3 10.0
T8: CP + CDB-6 + CDB-18 6.0 7.0 9.3
T9: CP + CDB-6 + CDB-11+ CDB-18 7.3 9.7 12.3
S.Em+ 0.47 0.31 0.29
CD at 1% 1.92 1.27 1.19
Note:
hill-1, respectively. However, combined inoculation of inoculants was superior and
significantly higher to other inoculations. Treatment T9 recorded a maximum number of
tillers hill-1 i.e. 12.3. The pots with the sole treatment of chlorpyrifos (T2) recorded the lowest
number of tillers hill-1 i.e. 5.0 while control showed 7.7 tillers hill-1.
4.8.1.3 Number of leaves per hill
The data revealing the effect of chlorpyrifos degrading bacteria on number of

leaves per hill of rice is presented in Table 14.
On 30th day of transplanting, the combined inoculation in treatment T9 (CP + CDB-

6 + CDB-11+ CDB-18) showed maximum number of leaves hill-1 i.e. 21.0 followed by
treatment T7 (CP + CDB-11 + CDB-18) with 19.7 leaves hill-1. The individual inoculation
of inoculants in treatment T3 (CP + CDB-6) showed of 13.7 leaves whereas treatment T4
(CP + CDB-11) and T5 (CP + CDB-18) recorded of 15.6 and 14.6 leaves
hill-1, respectively and these treatments were on par with each other. Combination of two
inoculants T6 and T8 recorded 18.3 and 17.3 leaves hill-1, respectively. The pots with the
sole treatment of chlorpyrifos (T2) recorded lower numbers of leaves hill-1 i.e. 9.3 compared
to control (16.3)

a maximum number of leaves hill-1 i.e. 42.3 followed by T7 which recorded 40.3 leaves hill-
1
. Combined inoculations were superior and significantly different when compared with
individual inoculation. The individual inoculation in treatments T3, T4 and T5 showed 33.3,
36.7 and 35.0 leaves hill-1, respectively; they were significant to each other. Combination of
two inoculants T6 and T8 showed 39.6 and 38.3 leaves hill-1, respectively. The pots with the
sole treatment of chlorpyrifos (T2) recorded a lower number of leaves hill-1 i.e. 26.3
compared to control (35.3).
On the 90th day of transplanting, the individual inoculation of inoculants in

treatments T3, T4 and T5 recorded 48.3, 52.3 and 49.7 leaves hill-1, respectively and they were
significant to each other. Similarly, combination of two inoculants T6, T7 and T8 showed
54.7, 56.3 and 53.7 leaves hill-1, respectively. However, combined inoculation of inoculants
was superior and significantly higher to other inoculations. Treatment T9 recorded a
maximum number of leaves hill-1 i.e. 58.6. The pots with the sole treatment of
Table 14: Number of leaves per hill of rice as influenced by the application of efficient
Number of leaves hill-1

Treatment
T1: Control 16.3 35.3 47.6
T2: CP 9.3 26.3 37.3
T3: CP + CDB-6 13.7 33.3 48.3
T4: CP + CDB-11 15.6 36.7 52.3
T5: CP + CDB-18 14.6 35.0 49.7
T6: CP + CDB-6 + CDB-11 18.3 39.6 54.7
T7: CP + CDB-11+ CDB-18 19.7 40.3 56.3
T8: CP + CDB-6 + CDB-18 17.3 38.3 53.7
T9: CP + CDB-6 + CDB-11+ CDB-18 21.0 42.3 58.6
S.Em+ 0.36 0.31 0.33
CD at 1% 1.50 1.27 1.35
Note:
chlorpyrifos (T2) recorded the lowest number of leaves hill-1 i.e. 37.3 while control showed
47.6 leaves hill-1.
4.8.1.4 Total dry weight
The data on influence of chlorpyrifos degrading bacteria on total dry weight of rice
is presented in Table 15.
The data revealed that, total dry weight at 30th, 60th and 90th DAT in T9 (CP + CDB-
6 + CDB-11 + CDB-18) recorded 2.94, 13.62 and 29.45 g hill-1, respectively. Individual
inoculations in treatment T3 (CP + CDB-6) recorded total dry weight of 2.15, 10.91 and
23.52 g hill-1 and T4 (CP + CDB-11) recorded 2.19, 11.52 and 25.32 g hill-1; whereas, T5 (CP
+ CDB-18) recorded 2.16, 11.29 and 24.57 g hill-1 at 30, 60 and 90 DAT, respectively.
Combination of two inoculants T6 (CP + CDB-6 + CDB-11) shown 2.45, 12.59 and 27.34 g
hill-1 and T8 (CP + CDB-180) recorded 2.56, 12.21 and 26.77 g hill-1; while, T7 (CP + CDB-
11 + CDB-18) recorded 2.67, 12.84 and 27.70 g hill-1 at 30th, 60th and 90th DAT, respectively.
These were significant to T2 at their respective days of recording. The pots with the sole
treatment of chlorpyrifos (T2) recorded 1.68, 9.25 and 20.22 g hill-1 at 30th, 60th and 90th DAT,
respectively.
4.8.2 Soil enzymatic analysis
4.8.2.1 Dehydrogenase activity
The dehydrogenase activity in soil was determined by the amount of TTC

(Triphenyl Tetrazolium Chloride) converted to TPF (Triphenyl Formazan) by the action of
the enzyme dehydrogenase. The amount of TPF formed was calculated from the standard
curve and the result was expressed as µg TPF formed per gram of soil per day (Table 16).
The data showed that, dehydrogenase activity of the soil was recorded highest on
60th DAT in T9 among all. Dehydrogenase activity at 30th, 60th and 90th DAT in T9 (CP +
CDB-6 + CDB-11 + CDB-18) recorded was 31.3, 54.0 and 44.7 µg TPF g-1 of soil d-1,
respectively. Individual inoculation in treatment T3 (CP + CDB-6) recorded dehydrogenase
activity of 21.3, 38.3 and 30.7 µg TPF g-1 of soil d-1, T4 (CP + CDB-11) recorded 24.6, 44.6
and 36.6 µg TPF g-1 of soil d-1, While, T5 (CP + CDB-18) recorded 22.3, 40.0 and 34.6 µg
TPF g-1 of soil d-1 at 30th, 60th and 90th DAT, respectively. Combination of two inoculants
Table 15: Total dry weight per hill of rice as influenced by the application of efficient
Total dry weight (g hill-1)

Treatment
T1: Control 2.11 11.04 22.25
T2: CP 1.68 9.25 20.22
T3: CP + CDB-6 2.15 10.91 23.52
T4: CP + CDB-11 2.19 11.52 25.32
T5: CP + CDB-18 2.16 11.29 24.57
T6: CP + CDB-6 + CDB-11 2.45 12.59 27.34
T7: CP + CDB-11 + CDB-18 2.67 12.84 27.70
T8: CP + CDB-6 + CDB-18 2.56 12.21 26.77
T9: CP + CDB-6 + CDB-11+ CDB-18 2.94 13.62 29.45
S.Em+ 0.05 0.04 0.13
CD at 1% 0.23 0.16 0.54
Note:
Table 16: Dehydrogenase activity of the soil as influenced by the application of
Dehydrogenase activity
Treatment (µg TPF g-1 d-1)
T1: Control 18.6 31.6 26.7
T2: CP 14.6 28.0 25.6
T3: CP + CDB-6 21.3 38.3 30.7
T4: CP + CDB-11 24.6 44.6 36.6
T5: CP + CDB-18 22.3 40.0 34.6
T6: CP + CDB-6 + CDB-11 26.6 47.6 40.3
T7: CP + CDB-11+ CDB-18 29.3 50.3 42.6
T8: CP + CDB-6 + CDB-18 26.0 45.6 38.6
T9: CP + CDB-6 + CDB-11+ CDB-18 31.3 54.0 44.7
S.Em± 0.31 0.27 0.33
CD at 1% 2.40 0.80 2.42
Note:
CP: Chlorpyrifos; CDB: Chlorpyrifos degrading bacteria; DAT: Days after
transplanting; Values are mean of three replications.
T6 (CP + CDB-6 + CDB-11) shown 26.6, 47.6 and 40.3 µg TPF g-1 of soil d-1 and T8 (CP
+ CDB-180) recorded 26.0, 45.6 and 38.6 µg TPF g-1 of soil d-1 while, T7 (CP + CDB-11+
CDB-18) recorded 29.3, 50.3 and 42.6 µg TPF g-1 soil d-1 at 30th, 60th and 90th DAT,
respectively. These were significant to T2 (CP as sole treatment) and T1 (control) at their
respective days of recording. T2 recorded 14.6, 28.0 and 25.6 µg TPF g-1 of soil d-1 at 30th,
60th and 90th DAT, respectively.
4.8.2.2 Phosphatase activity
The phosphatase activity in terms of concentration of p-nitrophenyl in each sample

was calculated by a standard curve of p-nitrophenol in water and was expressed as moles
of p-nitrophenol released per gram of dry soil per hour (Table 17).
The data revealed that phosphatase activity in the chlorpyrifos treated soils was
recorded highest on 60th DAT in T9 among all. Phosphatase activity at 30th, 60th and 90th
DAT in T9 (CP + CDB-6 + CDB-11 + CDB-18) recorded was 31.4, 41.8 and 35.9 µg PNP
g-1 of soil h-1, respectively. Individual inoculation in treatment T3 (CP + CDB-6) recorded
phosphatase activity of 21.8, 31.7 and 28.7 µg PNP g-1 soil h-1, T4 (CP + CDB-11) recorded
25.8, 35.7 and 31.8 µg PNP g-1 of soil h-1, While, T5 (CP + CDB-18) recorded 22.9, 34.8
and 30.8 µg PNP g-1 of soil h-1 at 30th, 60th and 90th DAT, respectively. Combination of two
inoculants showed increased effect compared to individual treatments where, T6 (CP +
CDB-6 + CDB-11) showed 28.4, 38.8 and 34.5 µg PNP g-1 of soil h-1 and T8 (CP + CDB-6
+ CDB-18) recorded 26.8, 37.9 and 33.9 µg PNP g-1 of soil h-1 while, T7 (CP + CDB-11 +
CDB-18) recorded 28.9, 39.9 and 34.9 µg PNP g-1 of soil h-1 at 30th, 60th and 90th DAT,
respectively. These were significant to T2 and T1 (control) at their respective days of
recording. T2 recorded 15.9, 25.7 and 22.7 µg PNP g-1 of soil h-1 at 30th, 60th and 90th DAT,
respectively.
4.8.3. Determination of microbial population in the soil
The soil samples were serially diluted and inoculated onto nutrient agar, Martin rose
Bengal agar and Kenknight & Muneer’s media plates, for the enumeration of bacteria,
fungi and actinomycetes, respectively. The number of colonies were represented as CFU
per gram of soil.
Table 17: Phosphatase activity of the soil as influenced by the application of efficient
Phosphatase activity
Treatment (µg PNP g-1 h-1)
T1: Control 21.1 27.8 25.7
T2: CP 15.9 25.7 22.7
T3: CP + CDB-6 21.8 31.7 28.7
T4: CP + CDB-11 25.8 35.7 31.8
T5: CP + CDB-18 22.9 34.8 30.8
T6: CP + CDB-6 + CDB-11 28.4 38.8 34.5
T7: CP + CDB-11+ CDB-18 28.9 39.9 34.9
T8: CP + CDB-6 + CDB-18 26.8 37.9 33.9
T9: CP + CDB-6 + CDB-11 + CDB-18 31.4 41.8 35.9
S.Em± 0.21 0.19 0.25
CD at 1% 2.71 2.47 2.40
Note:
4.8.3.1 Enumeration of bacteria
The data showed that bacterial population in the chlorpyrifos treated soils was
recorded highest on 60th DAT in T9 among all (Table 18).
The bacterial population at 30th, 60th and 90th DAT in T9 recorded was 43.3 × 106,
84.6 × 106 and 70.7 × 106 CFU g-1 of soil, respectively. Individual inoculation in treatment
T3 recorded 29.6 × 106, 67.7 × 106 and 51.0 × 106 CFU g-1 of soil, T4 recorded 32.6 × 106,
71.8 × 106 and 60.2 × 106 CFU g-1 of soil; while, T5 recorded 30.6 × 106, 70.0 × 106 and
55.7 × 106 CFU g-1 of soil at 30th, 60th and 90th DAT, respectively. Combination of two
inoculants in T6 shown 35.6 × 106, 76.7 × 106 and 64.1 × 106 CFU g-1 of soil and T8 recorded
34.6 × 106, 74.0 × 106 and 62.3 × 106 CFU g-1 of soil; while, T7 recorded 38.6 × 106, 79.8 × 106
and 68.2 × 106 CFU g-1 soil at 30th, 60th and 90th DAT, respectively. These were significant
to T2 (CP as sole treatment) and T1 (control) at their respective days of recording. T2
recorded 22.0 × 106, 55.0 × 106 and 38.3 × 106 CFU g-1 of soil at 30th, 60th and 90th DAT,
respectively.
4.8.3.2 Enumeration of fungi
The data showed that, the fungal population in the chlorpyrifos treated soils was
recorded highest on 60th DAT in T9 among all (Table 19)
The fungal population at 30th, 60th and 90th DAT in T9 recorded was 17.3 × 103, 24.3
× 103 and 21.0 × 103 CFU g-1 of soil, respectively. Individual inoculation in treatment T3
recorded 9.7 × 103, 17.6 × 103 and 13.6 × 103 CFU g-1 of soil, T4 recorded 12.7 × 103,
20.3 × 103 and 16.6 × 103 CFU g-1 of soil, while, T5 recorded 11.3 × 103, 18.6 × 103 and
14.6 × 103 CFU g-1 of soil at 30th, 60th and 90th DAT, respectively. Combination of two
inoculants T6 shown 14.6 × 103, 20.6 × 103 and 17.6 × 103 CFU g-1 of soil and T8 recorded 13.6
× 103, 21.0 × 103 and 16.6 × 103 CFU g-1 of soil; while, T7 recorded 15.6 × 103, 22.3 × 103 and
18.3 × 103 CFU g-1 soil at 30th, 60th and 90th DAT, respectively. These were significant to T2
(CP as sole treatment) and T1 (control) at their respective days of recording. T2 recorded 5.3
× 103, 13.0 × 103 and 8.6 × 103 CFU g-1 of soil at 30th, 60th and 90th DAT, respectively.
Table 18: Bacterial population of the soil as influenced by the application of efficient
Enumeration of bacteria
Treatments (× 106 CFU g-1 of soil)
T1: Control 26.6 63.0 48.6
T2: CP 22.0 55.0 38.3
T3: CP + CDB-6 29.6 67.7 51.0
T4: CP + CDB-11 32.6 71.8 60.2
T5: CP + CDB-18 30.6 70.0 55.7
T6: CP + CDB-6 + CDB-11 35.6 76.7 64.1
T7: CP + CDB-11+ CDB-18 38.6 79.8 68.2
T8: CP + CDB-6 + CDB-18 34.6 74.0 62.3
T9: CP + CDB-6 + CDB-11+ CDB-18 43.3 84.6 70.7
S.Em± 1.04 0.41 0.52
CD at 1% 4.26 1.22 3.33
Note:
Table 19: Fungal population of the soil as influenced by the application of efficient
Enumeration of fungi
Treatments (× 103 CFU g-1 of soil)
T1: Control 9.0 17.3 12.0
T2: CP 5.3 13.0 8.6
T3: CP + CDB-6 9.7 17.6 13.6
T4: CP + CDB-11 12.7 20.3 16.6
T5: CP + CDB-18 11.3 18.6 14.6
T6: CP + CDB-6 + CDB-11 14.6 20.6 17.6
T7: CP + CDB-11+ CDB-18 15.6 22.3 18.3
T8: CP + CDB-6 + CDB-18 13.6 21.0 16.6
T9: CP + CDB-6 + CDB-11+ CDB-18 17.3 24.3 21.0
S.Em± 0.31 0.29 0.35
CD at 1% 2.40 2.42 2.55
Note:
4.8.3.3 Enumeration of actinomycetes
The data revealed that, the population of actinomycetes in the chlorpyrifos treated
soils was recorded highest on the 60th DAT in T9 among all (Table 20).
The population of actinomycetes at 30th, 60th and 90th DAT in T9 recorded was 30.4
× 104, 45.5 × 104 and 40.4 × 104 CFU g-1 of soil respectively. Individual inoculation in
treatment T3 recorded 18.9 × 104, 31.7 × 104 and 27.7 × 104 CFU g-1 of soil, T4 recorded
22.9 × 104, 35.8 × 104 and 33.8 × 104 CFU g-1 of soil; while, T5 recorded 20.8 × 104,
34.8 × 104 and 30.5 × 104 CFU g-1 of soil at 30th, 60th and 90th DAT, respectively. Combination
of two inoculants T6 shown 24.8 × 104, 38.8 × 104 and 35.7 × 104 CFU g-1 of soil and T8
recorded 23.7 × 104, 36.9 × 104 and 34.8 × 104 CFU g-1 of soil; while, T7 recorded 25.8 × 104,
41.8 × 104 and 37.8 × 104 CFU g-1 soil at 30th, 60th and 90th DAT, respectively. These were
significant to T2 (CP as sole treatment) and T1 (control) at their respective days of recording.
T2 recorded 11.8 × 104, 17.5 × 104 and 14.8 × 104 CFU g-1 of soil at 30th, 60th and 90th DAT,
respectively.
4.8.4 Analysis of chlorpyrifos degradation in soil by the efficient bacterial isolates

using GC-MS/MS
The chlorpyrifos residue analysis was carried out using GC-MS/MS (make:
Shimadzu, model: TQ8030) (Plate 12) and the results are depicted in Table 21.
On the 30th day of CP application, the data indicated the major difference between
the treatments of individual inoculation and dual inoculations. The single inoculation in
treatment T4 (CP + CDB-11) showed maximum degradation i.e. 98.46 % with residual
concentration of 6.325 mg kg-1 followed by treatment T9 (CP + CDB-6 + CDB-11+
CDB-18) with 95.92 % degradation and residual concentration of 16.767 mg kg-1. The
individual inoculations in treatment T3 (CP + CDB-6) and T5 (CP + CDB-18) recorded
93.13 % and 94.11 % degradation and residual concentration of 28.214 mg kg-1 and 24.191
mg kg-1, respectively. Dual combination of inoculants in T6 (CP + CDB-6 +
CDB-11) registered 95.32 % degradation and the residual concentration was found to be
19.206 mg kg-1; whereas, T7 (CP + CDB-6 + CDB-1) and T8 (CP + CDB-6 + CDB-18)
showed 91.52 % and 89.89 % degradation and residual concentration of 34.812 mg kg-1
and 41.511 mg kg-1, respectively. The pots with the sole treatment of chlorpyrifos (T2)
Table 20: Population of actinomycetes in the soil as influenced by the application of
Enumeration of actinomycetes
Treatment (× 104 CFU g-1 of soil)
T1: Control 15.8 25.4 22.8
T2: CP 11.8 17.5 14.8
T3: CP + CDB-6 18.9 31.7 27.7
T4: CP + CDB-11 22.9 35.8 33.8
T5: CP + CDB-18 20.8 34.8 30.5
T6: CP + CDB-6 + CDB-11 24.8 38.8 35.7
T7: CP + CDB-11+ CDB-18 25.8 41.8 37.8
T8: CP + CDB-6 + CDB-18 23.7 36.9 34.8
T9: CP + CDB-6 + CDB-11+ CDB-18 30.4 45.5 40.4
S.Em± 0.20 0.39 0.29
CD at 1% 2.61 2.73 2.40
Note:
recorded 50.77 % reduction with 202.208 mg kg-1 of residual concentration. GC-MS/MS
spectrum of the standard as well as the samples are mentioned in the figures 3a-3b and 4a-
4h, respectively.
Table 21: Concentration and per cent degradation of chlorpyrifos by the efficient
bacterial isolates estimated using GC-MS/MS
Chlorpyrifos degradation analysis after 30

days of application
Treatment
Area Height Conc. CP Degradation
residue (%)
(mg kg-1)
Initial application (0th day) 158152

8933186 410.739 -
8
T1: Control - - BQL -
T2: CP 4397846 743565 202.208 50.77
T3: CP + CDB-6 613619 103105 28.214 93.13
T4: CP + CDB-11 137565 23153 6.325 98.46
T5: CP + CDB-18 526129 86769 24.191 94.11
T6: CP + CDB-6 + CDB-11 417723 67162 19.206 95.32
T7: CP + CDB-11+ CDB-18 757121 127449 34.812 91.52
T8: CP + CDB-6 + CDB-18 902826 156765 41.511 89.89
T9: CP + CDB-6 + CDB-11+ CDB-18 364662 61164 16.767 95.92

Note: Retention time = 19.774; m/z = 313.90>257.90; Limit of Quantification (LOQ) = 1 ppb;
BLQ = Below Quantification Level; CP: Chlorpyrifos
Y = 713.5075X - 745.4421
R^2 = 0.9988471 R = 0.9994234
Area(x10,000)
7.0 5
6.0 4
5.0
4.0
3
3.0
2.0
2
1.0
1
0.0
0 10 20 30 40 50 60 70 80 90 Conc.
Fig. 3a: Linearity calibration curve for 0.01 to 0.1 mg kg-1 of chlorpyrifos
Target 313.90>257.90 313.90>285.90 313.90>193.90
CHLORPYRIFOS
(x1,000)
313.90>257.90
20.067
313.90>285.90
1.50
313.90>193.90
1.25
1.00
0.75
0.50
0.25
19.8 19.9 20.0 20.1 20.2 20.3 20.4
Fig. 3b: Chromatogram of standard for 0.01 to 0.1 mg kg-1 of chlorpyrifos

(x100,000) (x100,000)
7.5 313.90>257.90 313.90>257.90
313.90>285.90 1.00 313.90>285.90
313.90>193.90 313.90>193.90
0.75
5.0
0.50
2.5
0.25
19.25 19.50 19.75 20.00 19.25 19.50 19.75 20.00
Fig. 4a: T2- Cp Fig. 4b: T3- Cp + CDB-6
(x10,000) (x10,000)
313.90>257.90 313.90>257.90
313.90>285.90 313.90>285.90
313.90>193.90 313.90>193.90
2.0 7.5
1.5
5.0
1.0
2.5
0.5
19.25 19.50 19.75 20.00 19.25 19.50 19.75 20.00
Fig. 4c: T4- Cp + CDB-11 Fig. 4d: T5- Cp + CDB-18

(x10,000) (x100,000)
7.0 313.90>257.90 313.90>257.90
313.90>285.90 1.25 313.90>285.90
6.0 313.90>193.90 313.90>193.90
1.00
5.0
4.0 0.75
3.0
0.50
2.0
0.25
1.0
19.25 19.50 19.75 20.00 19.25 19.50 19.75 20.00
Fig. 4e: T6- Cp + CDB-6 + CDB-11 Fig. 4f: T7- Cp+ CDB-11+ CDB-18
(x100,000) (x10,000)
313.90>257.90 313.90>257.90
1.50 313.90>285.90 6.0 313.90>285.90
313.90>193.90 313.90>193.90
1.25 5.0
1.00 4.0
0.75 3.0
0.50 2.0
0.25 1.0
19.25 19.50 19.75 20.00 19.25 19.50 19.75 20.00
Fig. 4g: T8- Cp + CDB-6+ CDB-18 Fig. 4h: T9- Cp + CDB-6 + CDB-11 + CDB-18
Fig. 4 (a-h): GCMS/MS Spectrum of chlorpyrifos from the soil samples at 50-dilution
Plate 12: GC-MS/MS instrument used in chlorpyrifos degradation analysis
Discussion
V. DISCUSSION
In India, rice is (Oryza sativa L.) is one of the important food grains and it is being
cultivated with an area of 43.9 million hectares. It is the staple food for most of the
population. Thus, maintenance of quality and quantity is a major task. Incidence of pests
on rice has been more frequent due to many environmental factors. Management of insect
pests often relies exclusively on synthetic insecticides, but indiscriminate use of pesticides
is common among farmers in developing countries. The production, distribution, use,
misuse and disposal or accidental spills of many agrochemicals have polluted some
environments to levels that threaten the health of humans, livestock, wildlife and indeed
whole ecosystems. Organophosphorus insecticides share about 80 % of the overall
agrochemicals in the world market and are used widely in agriculture. Chlorpyrifos is a
commonly used anti-cholinesterase organophosphate insecticide and it is toxic to a variety
of beneficial arthropods including bees, ladybird beetles and parasitic wasps.
The chlorpyrifos has been used to control gall midge (Orseolia oryzae), leaf-folder
(Cnaphalocrocis medinalis), leafhopper (Nephotettix virescens) and various insect pests.
Extensive use of chlorpyrifos contaminates air, ground water, rivers, lakes, rainwater and
fog water and it results in accumulation of pesticide residues in crops, soils, and biosphere
creating an ecological stress (Qiao et al., 2003). Prolonged exposure to chlorpyrifos results
in delayed seedling emergence, fruit deformities and abnormal cell division in plants
(Galloway and Handy, 2003).
The fate of this pesticide in the environment is associated with both abiotic and
biotic processes, including volatilization, photooxidation, chemical oxidation,
bioaccumulation, and microbial transformation (Liu et al., 2001). The half-life of
chlorpyrifos in soil generally ranges between 60-120 days (Howard, 1991).
The bioremediation is a transformation or degradation of contaminants into non-

hazardous or less hazardous chemicals. It describes several technologies and practices that
take advantage of natural systems and processes to clean up pollution. Bacteria are
generally used for bioremediation, but fungi, algae and plants have also been used.
Microbes that are present in pesticide contaminated sites for long duration, develops the
ability to degrade/tolerate such contaminant and such microbes with advanced/new traits
can be used for pesticide degradation (Farhan et al., 2012). Several chemicals have been
successfully removed from soil and aquatic environments using degrading microorganisms
such as parathion (Barles et al., 1979), coumaphos (Mulbry et al., 1998) and atrazine
(Struthers et al., 1998). In view of degradation of chlorpyrifos, the present investigation
was carried out for assessing the biodegradation efficiency of bacterial isolates isolated
from TBP command area.
The rhizosphere soil contains maximum microbial activity due to secretion of root
exudates by the plants. Soil samples were collected from top 10-15 cm of the soil profile
in the rhizosphere of paddy in sterilized polythene bags as the microbial population is
maximum in rhizosphere regions. The polythene bags were properly tied to avoid
contamination. The soil samples were preserved in a refrigerator to arrest biological
activity and to isolate the chlorpyrifos degrading bacteria.
5.2 Physico-chemical analysis of the soil samples
The pesticide degradation rates in the soil are a function of multiple factors
including population densities and activity of pesticides degrading microorganisms,
pesticide bioavailability and soil parameters such as pH, soil water content and
temperature (Parkin and Daniel, 1994). Analysis of soil has been extensively used in
agriculture and horticulture to assess soil health and provides useful information for
imposing effective soil and water management practices to boost the crop productivity.
The chemical analysis of soils, however, is of at most importance for adequate

and balanced nutrition of crops besides attaining the higher fertilizer use efficiency.
Assessment of soil characteristics such as soil pH is of paramount significance as it
enables to predict soil fertility, availability of nutrients and activity of microorganisms of
soil. Whereas electrical conductivity renders information about concentration of soluble
salts in soil and their effect on plant growth and may be soil microbes. Soil organic matter
measured as g kg-1. Organic carbon is of vital importance as it provides energy and body
building constituents for microbes in addition to many other functions. In the present
study physico-chemical analysis of soil samples collected from various locations revealed
a range of pH 6.95-8.88. Electrical Conductivity (EC) varied from 0.12-0.92 dS m-1;
while, percent organic carbon ranged from 3.18-6.75 % in forty soil samples.
5.3 Isolation and purification of the chlorpyrifos degrading bacteria from the
rhizospheric soils of rice
The chlorpyrifos degrading microbes can be isolated from various sources like
soil, corals, municipal waste, agrochemical industry waste and wastewater etc. The
perusal of literature has revealed that chlorpyrifos degrading microorganisms have been
extensively isolated from various sources like contaminated agricultural soils (Singh et
al., 2004; Yang et al., 2005), from sludge and waste water from pesticide manufacturing
units (Li et al., 2007; Latifi et al., 2012; Lu et al., 2013). In the present investigation
chlorpyrifos degrading bacterial isolations were carried out from soil samples collected
from different locations of TBP command area due to predominance of rice.
The collected soil sample were serially diluted and plated on MSM media
amended with 200 ppm of chlorpyrifos and the plates were incubated at room temperature
(30 ± 1 °C) for 7 days. Totally, 25 chlorpyrifos degrading bacteria were isolated
considering the profused growth on MSM media plates supplemented with 200 ppm
chlorpyrifos.
Similar study was conducted by Sonali and Tushar (2017). Two bacterial strains
from rice soils were isolated that were capable of utilizing chlorpyrifos as a sole source of
carbon and energy for growth viz., TB251 and TB252. These bacteria were further studied
for identification and characterization.
Deepti et al. (2015) isolated two chlorpyrifos degrading bacteria using serial
dilution technique followed by selective enrichment on minimal medium with chlorpyrifos
as carbon source; from soil samples collected from a sugarcane field.
Bhagobaty and Mallick (2008) isolated four bacterial strains belonging to

Pseudomonas genera from wastewater irrigated agricultural soil with a previous history of
chlorpyrifos use.
5.4 In vitro screening for growth of chlorpyrifos degrading bacterial isolates in

minimal salt medium by enrichment culture method
The selective secondary screening of 25 chlorpyrifos degrading bacterial isolates

was carried out in MSM media supplemented with chlorpyrifos as sole source of carbon.
A loop full of 24 hr old test organism was inoculated into 250 ml conical flak containing
100 ml of pre-sterilized minimal salt media supplemented with different concentrations of
CP and incubated on orbital shaker for a week time at 150 rpm.
The aliquot of the culture was examined for the development of turbidity using
spectrophotometer at OD600 and 10 µl of aliquot was plated on the nutrient agar plates to
confirm the growth of isolates. Out of 25 isolates, eleven isolates grew on the media
supplemented with 300 ppm CP, nine isolates showed positive growth on the media
supplemented with 400 ppm CP and finally, seven isolates were able to grow in the media
supplemented with 600 ppm CP.
Similar work was reported by Bhagobaty and Mallick (2008). Four chlorpyrifos
degrading bacteria were isolated and screened from waste water and irrigated agricultural
soils on minimal salt media supplemented with chlorpyrifos, which could grow at higher
concentration of chlorpyrifos (3200 µg ml-1) in the media.
Similarly, other workers Ahemad and Khan (2011) studied on assessment of the
pesticide tolerance of plant growth promoting rhizobacteria recovered from the
rhizosphere of four crops (mustard, chickpea, green gram and lentil). They isolated a total
of 53 rhizobacteria belongs to the genera viz., Pseudomonas, Enterobacter and Klebsiella
which are capable of tolerating pesticides in the range of 400-3200 µg ml-1.
5.5 Estimation of percent chlorpyrifos degradation by the efficient isolates under

in vitro conditions
The chlorpyrifos degrading capacity of the isolates was measured at an interval of two days
for a span of ten days using UV spectrophotometer. After 10 days, total degradation of
chlorpyrifos by all the isolates ranged between 50 % to 72.4 %. Highest degradation was
showed by CDB-11 with 72.4 % followed by the isolates CDB-18 and CDB-6 which
showed chlorpyrifos degradation up to 70.2 % and 68.5 %, respectively (Fig 5a-5b). This
is due to utilization of the supplemented chlorpyrifos (200 ppm) as their carbon and energy
source for the growth in the media.
Similar work was carried out by Sharma et al. (2016). Bacillus and Micrococcus sp.
were isolated from chlorpyrifos contaminated soils. The species were tested for their
capabilities to degrade the pesticide at two different concentrations (0.05 % and 0.1 %)
Concentration of chlorpyrifos (ppm)
Concentration of Cp (ppm)
Initial
200
2nd day
180
160 4th day
140
120 6th day
100
8th day
80
60 10th day
40
20
0
CDB-1 CDB-6 CDB-11 CDB-14
Isolate CDB-16 CDB-18 CDB-19
Fig. 5a. Residual concentration of chlorpyrifos at 200 ppm concentration in MSM broth inoculated with efficient bacterial isolates
Percent degradation of chlorpyrifos by the effecient isoates
80
70
60
Degradation (%)
50
Initial
2nd day
40
4th day
6th day
30
8th day
10th day
20
10
0
CDB-1 CDB-6 CDB-11 CDB-14 CDB-16 CDB-18 CDB-19
Isolate
Fig. 5b. Degradation percentage of chlorpyrifos at 200 ppm concentration in MSM broth inoculated with efficient
bacterial isolates
spectrophotometrically. After 10 days of incubation, maximum degradation of chlorpyrifos
was observed in Micrococcus sp. with 0.1 % of pesticide which was observed as 71.6 %.
Similarly, Hamsavathani et al. (2017) screened two pesticide degrading bacteria for
the degradation of chlorpyrifos in MSM broth supplemented with 0.5 % chlorpyrifos and
monitored every 48-72 hrs in spectrophotometer. The degradation efficiency of the strains
was determined and estimated by the removal percentage of chlorpyrifos from the liquid
culture. S. aureus was more potent in degrading the 80 % of the total compound from the
media in 2 weeks of incubation
Jayamadhuri and Ramaswamy (2009) isolated bacterial cultures by selective

enrichment technique. Species of Bacillus and Pseudomonas were tested for their ability to
degrade 75 % of chlorpyrifos in mineral salts medium within 7 days of incubation. Carbon
source plays a major role in microbial nutrition and growth. Bacteria utilises chlorpyrifos
as sole carbon source for its growth when supplemented in MSM media.
5.6.1 Morphological characterization of the efficient chlorpyrifos degrading bacterial

isolates
Both morphological as well as biochemical characterization technologies have

been immensely useful and applied in carrying out characterization of most of the
microorganisms. In the present study different morphological characters viz., colour, size,
texture and elevation of the colonies along with microscopic characters such as gram
reaction, shape and motility were studied. It was observed that out of the total CP degrading
isolates, 63 % were gram negative in nature whereas only 37 % were found to be gram
positive. 18 % of the isolates were nonmotile while, remaining 72 % of the isolates were
motile and all were found to be rod shaped.
In literature also variations in gram character, shape, arrangement, motility and

spore formation have been reported. Savitha and Saraswati (2012) recorded gram positive,
motile and endospore forming bacteria for chlorpyrifos degradation.
Farhan et al. (2012) reported colonies of Pseudomonas strain WW5 as gram

negative, rod shaped and motile in nature.
5.6.2 Biochemical characterization of the efficient chlorpyrifos degrading bacterial
isolates
The biochemical characterization of eleven chlorpyrifos degrading bacterial isolates

revealed that all chlorpyrifos degrading bacterial isolates were found positive for starch
hydrolysis, catalase activity, citrate utilization, gas production and denitrification tests.
Whereas negative for urease test, methyl-red test, and indole test and variation was
observed in H2S production, gelatin liquefication, Voges-Proskauer test as well as casein
hydrolase test.
All the 11 efficient isolates showed positive results for starch hydrolysis test. In this
test, petri-plates containing starch agar were inoculated with test cultures and incubated for
three days. Then they were flooded with Lugol’s iodine solution and the black background
was observed due to the formation of starch iodine complex, clear zone around the colony
indicates that the starch has degraded due to the production of amylase and in this
investigation, there was a clear zone around the colonies after the addition of iodine and
reported as positive for the test was considered as positive for the test.
All the 11 isolates showed positive results for catalase test. In this test, nutrient
agar slants were inoculated with test organisms and incubated at 30 °C for 24 hours. After
incubation the tubes were flooded with one ml of three percent hydrogen peroxide and
observed for production of gas bubbles. Since all the isolates showed the occurrence of
gas bubbles indicating the presence of catalase enzyme and they were scored positive for
catalase activity.
None of the 11 isolates showed positive results for urease activity. In this study,
cultures were inoculated in to five ml of pre-sterilized urea broth containing phenol red
as pH indicator. The tubes were incubated for 48 hours at 30 °C. But no isolates showed
development of pink color.
All the 11 isolates showed negative results for methyl red test. In the present
context, the test tubes containing pre-sterilized MR-VP broth were inoculated with the
test cultures. The tubes were incubated for 48 hours. Then, five drops of methyl red
indicator were added to each tube and gently shaken. The production of red color was
taken as positive for the test and production of yellow color was taken as negative for the
test. All the isolates showed yellow color at the end of the test.
Three isolates tested positive and remaining eight isolates showed negative results
for Voges-Proskauer test. In the present study, test cultures were inoculated to the pre-
sterilized tubes containing MR-VP broth. The tubes were incubated for 48 h. After
incubation ten drops of Barritt’s reagent A was added and gently shaken followed by
addition of ten drops of Baritt’s reagent B. The development of rose color was taken as
positive for the test.
All the 11 isolates showed positive results for citrate utilization test. In this test,
the isolates were streaked on Simmon’s citrate agar slants and incubated at 28 ± 2 °C for
24 h. Change in color from green to blue occurs as bacteria metabolize citrate,
the ammonium salts are broken down to ammonia, which increases alkalinity. The shift
in pH turns the bromothymol blue indicator in the medium from green to blue.
Two isolates showed negative for the test, remaining nine isolates showed positive
results for gelatin liquification test. In the present context, the test cultures were
inoculated to the pre-sterilized nutrient gelation deep tubes, and tubes were incubated at
28 ± 2 °C for 24 h. Following this, the tubes were kept in a refrigerator at 4° C for 30
minutes. Those tubes which get solidified on refrigeration were taken as negative for the
test. As gelatin remains solid below 22 °C while the degraded form of gelatin i.e. amino
acids and peptides is to remain liquid below 22 °C. Thus, the tubes with cultures that
remained liquefied were taken as positive.
Four isolates showed negative results, remaining seven isolates showed positive
results for casein hydrolysis test. In this study, the plates containing skim milk agar were
streaked with test cultures and incubated at 30 °C for one week. The clear zone around the
colony was observed against creamy white background. This is because, casein imparts
white color to the media, which up on degradation by the caseinase enzyme, media loses
color and becomes hallow. Thus, colonies with hallow zones were scored as positive
All the 11 isolates produced gas in the glucose broth and nitrate broth thus showed
positive results for both gas production and denitrification test.
Three isolates tested positive and remaining eight isolates showed negative results
for H2S production and all the 11 isolates showed negative results for indole test. For these
tests, the bacterial isolates were inoculated into test tubes containing 5 ml of sterile SIM
agar medium and were incubated at 28 °C. The formation of a black ring in the medium
due to conversion of ferrous sulfate to ferrous sulfide was taken as positive for H2S
production. For indole test, each tube was added with 10 drops of Kovac’s reagent. There
was no change in color as there was no breakdown of tryptophan into amino group and
indole.
Similar results were obtained by Bhagobaty and Mallick (2008). Four bacterial
isolates belonging to the genus Pseudomonas were reported, which tested positive for
oxidase, negative for indole. Two isolates, RA-3 and RA-20 showed negative for methyl
red and only RA-5 was found positive for Voges-Prosekeur test.
Similarly, Dilfuza (2005) isolated organisms from the rhizosphere of different crops
and identified them as Pseudomonas species based on the biochemical characterization. A
Pseudomonas strain showed positive results for gelatine liquefaction, citrate utilization,
oxidase, catalase tests and it showed negative results for casein hydrolysis and urease tests.
5.7 In vitro screening of efficient chlorpyrifos degrading bacteria for their plant
growth promoting attributes
The efficient chlorpyrifos degrading isolates were selected after screening for their
capacity to degrade higher amount of chlorpyrifos and mechanisms which could render
promotion of growth of the plants. These bacteria are plant root colonizers and some
organisms might be deleterious for the crops. Screening for the beneficial traits is a pre-
requisite for selecting the potential bioinoculant for plant. Many Pseudomonas and Bacillus
isolates were known to produce different plant growth promoting substances like indole
acetic acid, siderophore and solubilize inorganic phosphate. These enhance plant growth
by direct and indirect mechanisms. The production of phytohormones and other
sequestering substances lead to enhancement and proliferation of the roots indirectly
increasing the growth of the plants.
When the chlorpyrifos degrading bacterial isolates grown in culture medium

supplemented with tryptophan, the isolates produced IAA as detected by the Salkowaski’s
reagent under spectrophotometer at OD530. All the isolates produced IAA and it ranged
between 10.59 μg ml-1 and 20.10 μg ml-1. Significantly higher concentration of IAA was
observed by CDB-11 (20.10 μg ml-1) which is followed by CDB-18 (18.51 μg ml-1)
(Fig. 6).
Similar results were observed by Reetha et al. (2014). Pseudomonas sp and Bacillus
sp were isolated from rhizosphere of onion and analyzed for the production of IAA under
in vitro and also studied the effect of these bacteria on plant growth of onion plant.
Using Chrome Azurol S (CAS) reagent, all the eleven isolates were quantified using
CAS shuttle assay of Payne (1994). The values ranged between 51.33 % and 64.33 %. The
isolate CDB-11 produced significantly higher siderophore of 64.33 % followed by the
isolate CDB- 18, that produced 61.33 %. This character of iron chelation is an important
factor for the rhizobacteria, as it deprives the pathogen from the available iron in the
surrounding (Fig. 7).
Similar work was reported by Sreedevi et al. (2014). Ten Pseudomonas spp. were isolated
from paddy soil. Among them, three Pseudomonas isolates P1, P2 and P3 showed
siderophore production on Chromo-azural S agar plate medium. Maximum siderophore
production was observed to be 94, 88 and 83 % for Pseudomonas 1, Pseudomonas 2 and
Pseudomonas 3 isolates, respectively.
Similarly, Tailor and Joshi (2012) isolated seven bacterial isolates from sugarcane
rhizosphere. All the isolates were found to produce more than 85 % siderophore units.
Among them, S-11 was found be the most efficient siderophore producer (96 % SU) which
was identified as Pseudomonas sp.
All the eleven bacterial isolates were able to solubilize phosphate on Pikovskaya’s
media containing Tri calcium phosphate. The solubilization zone diameter was in the range
of 8.3 mm to 17.6 mm. Among eleven bacterial isolates, CDB-18 showed the highest
solubilization zone (17.66 mm) with an efficiency of 132.48 % followed by CDB-11 (16.66
mm) which showed 135.33 % efficiency (Fig. 8).
Similar work was carried out by Fekadu (2013). Twelve Pseudomonas strains from
rhizospheric soil of Faba bean were isolated and tested for phosphate solubilization. All
IAA (μg ml-1)
25
20
Conc. (μg ml-1)
15
10
0
CDB-1 CDB-2 CDB-3 CDB-6 CDB-11 CDB-12 CDB-14 CDB-16 CDB-17 CDB-18 CDB-19
Isolate
Fig. 6. IAA production by efficient chlorpyrifos degrading bacterial isolates

Siderophore units (%)
70
60
Siderophore units (%)
50
40
30
20
10
0
CDB-1 CDB-2 CDB-3 CDB-6 CDB-11 Isolate
CDB-12 CDB-14 CDB-16 CDB-17 CDB-18 CDB-19
Fig. 7. Siderophore production by efficient chlorpyrifos degrading bacterial isolates

tested isolates of Pseudomonas strains had a potential of phosphate solubilization on
Pikovskaya’s media and summarized that all Pseudomonas strains can be used as bio-
fertilizers for soil fertility improvement.
Similarly, Manouchehr et al. (2016) studied the phosphate solubilization potential

of inhabiting bacteria from rhizosphere of Avicennia marina plants. Among 13 isolates
screened, four high potential solubilizing bacteria were selected based on the formation and
dimension of clear halo zone around the colonies. The isolates were found to be Bacillus,
Pseudomonas and Acinetobacter species.
5.8 Evaluation of efficient chlorpyrifos degrading bacteria on the growth of

paddy under pot culture
The three efficient bacterial isolates capable of degrading chlorpyrifos which fared
best for chlorpyrifos degradation capability as well as growth promotion attribute
conditions viz., CDB-6, CDB-11 and CDB-18 were evaluated individually and in
combination for their effect on growth characteristics of rice crop under pot culture
conditions supplemented with 0.05 % of chlorpyrifos.
5.8.1 Influence on the plant growth parameters
Significant increase in the plant height and number of tillers/hills was noticed at
different growth stages of crop (30th, 60th and 90th DAT) as influenced by efficient
chlorpyrifos degrading bacteria.
On the 30th day after transplanting, there was a significant difference between
combined inoculation of three chlorpyrifos degrading bacterial isolates when compared to
treatments with individual inoculation. The combined inoculation in treatment T9 (CP +
CDB-6 + CDB-11 + CDB-18) showed maximum plant height of 29.45 cm and 7.3 number
of tillers hill-1. Which is followed by treatment T7 (CP + CDB-11 + CDB-18) with 27.50
cm and 6.6 number of tillers hill-1. The pots with sole treatment of chlorpyrifos (T2)
recorded lower plant height of 18.49 cm and 3.7 tillers hill-1 compared to control
(24.67 cm and 5.0, respectively). The same trends were observed at 60th and 90th DAT
(Fig. 9 & 10).
Phosphate solubilization
20 160
18
140
Solubilization effeciency (%)

16
120
14
Diameter (mm)
100
12
10 80
8
60
6
40
4
20
2
0 Isolate 0
CDB-1 CDB-2 CDB-3 CDB-6 CDB-11 CDB-12 CDB-14 CDB-16 CDB-17 CDB-18 CDB-19
Solubilization zone (mm) Culture diameter (mm) Solubilization efficiency (%)
Fig. 8. Phosphate solubilization by efficient chlorpyrifos degrading bacterial isolates

Thirty days after transplanting, the data indicated that there was a significant
difference between combined inoculation of three isolates compared to treatments of
individual inoculation with respect to number leaves hill-1. The combined inoculation in
treatment T9 showed maximum number of leaves hill-1 i.e. 21.0 followed by treatment T7
(CP + CDB-11 + CDB-18) with 19.7 leaves hill-1. The pots with sole treatment of
chlorpyrifos (T2) recorded lower numbers of leaves hill-1 i.e. 9.3 compared to control (16.3).
Same trend was followed at 60th and 90th days after transplanting (Fig. 11).
The data revealed that, total dry weight at 30th, 60th and 90th DAT in T9 (CP + CDB-6 +
CDB-11 + CDB-18) recorded 2.94, 13.62 and 29.45 g hill-1, respectively. Among individual
inoculations, treatment T4 (CP + CDB-11) was recorded highest i.e. 2.19, 11.52 and 25.32
g hill-1 at 30th, 60th and 90th DAT, respectively. Among combination of two inoculants, T7
(CP + CDB-11+ CDB-18) recorded 2.67, 12.84 and 27.70 g hill-1 at 30th, 60th and 90th DAT,
respectively. These were significant to T2 at their respective days of recording. The pots
with sole treatment of chlorpyrifos (T2) recorded 1.68, 9.25 and 20.22 g hill-1 at 30th, 60th and
90th DAT, respectively (Fig. 12).
Different pesticides have been found to have adverse effects on plant growth
parameters such as plant height, weight and root nodulation. The negative effect of
pesticides on plant growth can be attributed to inhibition of electron flow in the
photosynthetic chain. OPs and DDT have been found to affect photosynthesis by
uncoupling the photosynthetic electron flow such that ATP synthesis is blocked
(Tiyagi et. al, 2004). Enhanced plant (O. sativa) growth was observed when soils were
inoculated with CP degrading bacterial strains. Following inoculation with CDB-6,
CDB-11 and CDB-18, increase in plant growth was monitored by tracking parameters. The
enhanced rate of plant growth can be attributed to IAA production, phosphate solubilization
and decrease in toxicity of pesticides owing to its increased degradation.
Similar findings were observed by Akbar and Sikandar (2016). Influence of

bacterial presence on plant growth and pesticide degradation was studied using plant
growth experiments taking cowpea as test crop. CP addition to soil affected a reduction in
certain plant parameters such as % germination (14.2 %), shoot length (2 cm), shoot fresh
weight (0.29 g), root fresh weight (0.05 g), shoot dry weight (0.2 g) and root dry weight
Plant height (cm)
90
80
70
Plant height (cm)
60
50
40
30
20
10
Treatment
0
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Fig.9. Plant height of rice as influenced by the application of efficient chlorpyrifos degrading bacterial inoculants
14
12
10
Treatment
0
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Fig.10. Number of tillers per hill of rice as influenced by the application of efficient chlorpyrifos degrading bacterial inoculants
Number of leaves hill-1
Number of leaves hill-1 60
50
40
30
20
10
0 Treatment
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Fig. 11. Number of leaves per hill of rice as influenced by the application of efficient chlorpyrifos degrading bacterial inoculants
Total dry weight (g hill-1)
Total dry weight (g hill-1) 30
25
20
15
10
Treatment
0
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Fig. 12. Total dry weight per hill of rice as influenced by the application of efficient chlorpyrifos degrading bacterial inoculants
(0.02 g). Plants grown in CP supplemented soils inoculated with CP-degrading bacterial
strains exhibited significant enhancement in growth in terms of height and weight. An
increase of 6.5 and 7.8 cm in shoot length and of 12.1 and 7.3 cm in root length was
observed in case of JCP4 and FCP1, respectively.
Similarly, Bhagat et al. (2017) conducted pot assay to check the efficacy of the two
best isolates for pesticide degradation and growth of Capsicum annuum plant. There was
significant increase in the height of plant and size of leaves when plants were grown in
pesticide contaminated soil in presence of isolates S1-5 and S1-6, separately. The pesticide
degradation by these two isolates was confirmed using gas chromatography technique.
5.8.2 Estimation of soil enzymatic activities
The data showed that, activity of the dehydrogenase enzyme was recorded highest on 60th
DAT in T9 among all. Dehydrogenase activity at 30th DAT in T9 (CP + CDB-6 + CDB-11
+ CDB-18) recorded was 31.3 µg TPF g-1 of soil d-1. T2 recorded lowest activity of 14.6 µg
TPF g-1 of soil d-1 compared to control (18.6 µg TPF g-1 d-1). Same trend was followed at
60th and 90th days after transplanting (Fig. 13).
Whereas the phosphatase activity in terms of concentration of p-nitrophenyl in each

sample was calculated by a standard curve of p-nitrophenol in water and was expressed as
mole of p-nitrophenol released per gram of dry soil per hour. Phosphatase activity at 30
DAT in T9 recorded was 31.4 µg PNP g-1 h-1 of soil. T2 recorded 15.9 µg PNP g-1 h-1 of soil,
which was the lowest among all. The activity increased in all the treatments up to 60th DAT
and then reduced slightly at 90th day (Fig. 14).
The data showed that the untreated soil with chlorpyrifos gave higher values of
enzymatic activity as compared with the soil treated with the pesticide. The results obtained
was due to the fact that pesticides application to the soil inhibits the activities of different
soil microorganisms. This result agrees with that obtained by Balinove et al. (1997) and
Lan et al. (2006). It was reported that, pesticides application to the soil had adverse effect
on microbial populations and consequently the microbial enzyme activities were decreased.
In addition, the soil treated with chlorpyrifos and inoculated with the mixture of the
tested bacteria showed higher enzymatic activity than the soil inoculated with each one
individually. Higher values of enzymatic activity in case of the soil inoculated with the
Dehydrogenase activity (µg TPF g-1 d-1)
60
Dehydrogenase activity (µg TPF g-1 d-1)
50
40
30
20
10
0 Treatment
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Fig.13. Dehydrogenase activity of the soil as influenced by the application of efficient chlorpyrifos degrading bacterial inoculants
Phosphatase activity (µg PNP g-1 h-1)
45
Phosphatase activity (µg PNP g-1 h-1)
40
35
30
25
20
15
10
Treatment
0
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Fig. 14. Phosphatase activity of the soil as influenced by the application of efficient chlorpyrifos degrading bacterial inoculants
mixture of the strains is likely due to the synergistic effect between the strains. Similar
result was observed by Aislabie and Jones (1997). It was mentioned that, some pesticides
are readily degraded by microorganisms including members of genera Alcaligenes,
Bacillus, Flavobacterium, Pseudomonas, Streptomyces and Rhodococcus.
5.8.3 Determination of microbial population in the soil
The soil samples were weighed, serially diluted and inoculated onto Nutrient agar,
Martin rose Bengal agar and Kenknight & Muneer’s media plates, for the enumeration of
bacteria, fungi and actinomycetes, respectively.
The results revealed that the soils treated with bacterial inoculants showed increased
microbial population on 30th day after inoculation compared to soil that was solely treated
with chlorpyrifos. The same trend was followed on 60th day as well. The pot soils with
chlorpyrifos as sole treatment resulted in lesser population of all the three microflorae viz.,
bacteria, fungi and actinomycetes compared to the control (Fig. 15, 16 and 17). The
population of the microbes in the control is due to the extraneous inoculation through the
irrigation water, FYM, etc.
There have been many contradictory reasons for the change in the microbial
population due to chlorpyrifos application. Researchers have reported short term inhibitory
effect on the total bacterial population (Pandey and Singh, 2004). On the other hand, some
studies showed significant increase in the same after chlorpyrifos treatment due to
application of chlorpyrifos degrading bacteria (Rani et al., 2008).
5.8.4 Estimation of chlorpyrifos degradation in the soil by the efficient bacterial

isolates using GC-MS/MS
On 30th day of chlorpyrifos (CP) application, the data indicated that comparatively
higher degradation was found in T4 (98.46 %) followed by T9, that showed degradation of
95.92 %. Close results were observed in treatments T3 and T5, which showed 93.13 % and
94.11 % of degradation. The pots with sole treatment of chlorpyrifos (T2) recorded
50.77 % reduction (Fig. 18).
The enhancement in the rate of CP degradation in soil could be attributed to the

greater pesticide degrading activity of the inoculated microbes. Plant roots secrete exudates
that can serve as a source of nutrients for microbial growth which in turn degrades pesticide
at an accelerated rate. In several studies, the plant seedlings have been shielded from the
toxic effects of various pesticides by bio-augmenting the soil with pesticide degrading
bacteria. Co-inoculations showed slightly lower degradation when compared to T4. This
might be due to competition between the organisms for establishment in the rhizosphere
and time lapse between the establishment and metabolism. Establishment of synergism
between the organisms becomes critical step in rapid degradation which is impeded by
initial competition. Whereas in T2, the degradation might be due to phytoremediation and
various nonbiological factors viz., hydrolysis, photo-remediation, as well as volatilization
losses.
Enumeration of actinomycetes (× 10⁴ CFU g⁻¹ of soil)
Population of actinomycetes 50
45
(× 10⁴ CFU g⁻¹ of soil)
40
35
30
25
20
15
10
0
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Treatment
Fig. 17. Population of actinomycetes in the soil as influenced by the application of efficient chlorpyrifos degrading
bacterial inoculants
Enumeration of bacteria (× 106 CFU g-1 of soil)
90
80
(×106 CFU g-1 of soil)
Bacterial population
70
60
50
40
30
20
10
0
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Treatment
Fig. 15. Bacterial population of the soil as influenced by the application of efficient chlorpyrifos degrading bacterial inoculants
Enumeration of fungi (× 103 CFU g-1 of soil)
25
20
(× 103 CFU g-1 of soil)
Fungul population
15
10
0
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Treatment
Fig. 16. Fungal population of the soil as influenced by the application of efficient chlorpyrifos degrading bacterial inoculants
Chlorpyrifos degradation analysis usinag GC-MS/MS
250 120
Chlorpyrifos conc. (mg kg⁻¹)
100
200
Degradation (%)
80
150
60
100
40
50
20
Treatment
0 0
T₁ T₂ T₃ T₄ T₅ T₆ T₇ T₈ T₉
Conc. CP residue (mg kg⁻¹) % Degradation
Fig. 18. Chlorpyrifos degradation analysis using GC-MS/MS after 30 days of application
Similar findings were observed by Akbar and Sikandar (2016). Two bacterial strains JCP4
and FCP1, exhibiting chlorpyrifos-degradation potential were isolated from pesticide
contaminated agricultural fields. GC-MS analysis of the soil revealed that these isolates
were able to degrade 84.4 % and 78.6 % of the initial concentration of chlorpyrifos.
Combined use of plants and microbes for chlorpyrifos biodegradation was

investigated by Ahmad et al. (2012). The plant and microbes used were rye grass and
Bacillus pumilis. The combination of plant and microbes degraded 97 % of chlorpyrifos
within 45 days in soil experimentation. The fact that exogenous microbes could be used
successfully for the bioremediation process was highlighted by the study.
Similar work was carried by Vidyalakshmi et al. (2009). Three aerobic bacterial
consortia, AC, BC and DC were developed from four isolates viz., Pseudomonas
aeruginosa, Bacillus cereus, Klebsiella sp., and Serratia marscecens which were able to
degrade chlorpyrifos in soil after 30 days by 50 %, 56 %, and 64 %, respectively.
Summary and Conclusions

VI. SUMMARY AND CONCLUSIONS
The study entitled “A study on chlorpyrifos degrading bacteria under rice

ecosystem” was conducted in Department of Agricultural Microbiology, University of
Agricultural Sciences, Raichur as well as in Pesticide Residue and Food Quality Analysis
Laboratory, UAS Raichur, Karnataka. Prime importance was given to the degradation of
chlorpyrifos both in vitro as well as in vivo.
Two major rice growing districts in Karnataka viz., Raichur and Koppal were
surveyed and selected for sample collection. Fourteen sites were selected from both the
districts so that a minimum of 10 km distance was maintained between each. Soil samples
collected from each location had a history of continual and extensive exposure to
chlorpyrifos for 2 decades.
Forty soil samples were collected, stored and analyzed for physicochemical
properties. pH and electrical conductivity (EC) of these samples ranged between 6.95-8.8
and 0.12-0.92 dSm-1, respectively; whereas per cent organic carbon (% OC) was found to
be in the range of 3.92-6.75 %.
Twenty-five bacterial isolates were isolated from a total of 40 soil samples by

enrichment technique using MSM agar supplemented with 200 ppm chlorpyrifos. The
colonies exhibiting profused growth on the plates after 7 days of incubation at 37 °C were
selected and purified for future investigation.
Secondary screening was carried out for all the twenty-five isolates in minimal salt
media using chlorpyrifos as the sole source of carbon. Initial screening was carried out with
300 ppm CP in the media where 11 isolates were able to grow and increase the turbidity of
the media. Further, the concentration of CP was increased to 400 ppm and it was observed
that 9 isolates showed positive growth. Finally, the isolates were screened for 600 ppm
where 7 isolates slightly increased the turbidity of the media showing a positive growth
response after 7 days of incubation at 37 °C.
The seven efficient isolates were analysed for in vitro degradation of chlorpyrifos
using UV- spectrophotometer at 200 ppm CP concentration in 100 ml minimal salt media,
which revealed that, CDB-18, CDB-11 and CDB-6 showed 70.2 %, 72.4 % and 68.5 % CP
degradation, respectively at 37 °C by the end of 10th day and they were found to be efficient
in degrading chlorpyrifos.
These isolates were able to utilize chlorpyrifos as a carbon source for their growth
and metabolism indicating the biodegradation of the chlorpyrifos by the isolates.
Various morphological characters viz., color, size, texture, elevation of the colonies
and microscopic characters viz., gram reaction, shape and motility were studied for eleven
bacterial isolates.
The observations revealed that the colonies of the isolates differ morphologically.
All the isolates were rod-shaped, 8 isolates were gram-negative and the remaining 3 were
gram-positive. These isolates were subjected for the biochemical characterization. The
results proved that the isolates were positive to catalase, starch hydrolysis, nitrate reduction,
oxidase test, citrate utilization and gas production and negative for urease, indole and
methyl red test. Based on the test results, isolates were identified as Bacillus spp and
Pseudomonas spp according to specific characters described in Bergey′s Manual of
Systematic Bacteriology (1994).
The efficient isolates were studied for their mechanisms in plant growth promotion
such as the production of siderophores, IAA and in vitro phosphate solubilization. All the
isolates were scored positive for the qualitative test for beneficial traits.
CDB-11 recorded a maximum siderophore production of 64.33 % followed by the

isolates CDB-6 and CDB-18 which produced 61.33 % and 59.66 % siderophore,
respectively. The significantly higher concentration of IAA was recorded by CDB-11
(20.10 μg ml-1) followed by CDB-18 (18.51 μg ml-1). Similarly, highest phosphate
solubilization efficiency was recorded by CDB-11 (135.11 %) followed by CDB-18
(132.48 %). In all the tests for beneficial traits, CDB-11 was superior among the other
isolates.
These three isolates were further tested under pot culture conditions to assess their
chlorpyrifos degrading activity in the soil at 500 ppm CP concentration and their effect on
the growth attributes of rice. Among all the treatments, rice seedlings treated with
combination of all three isolates (CDB-6, CDB11 and CDB-18) recorded a significant
increase in the growth parameters at 90th DAT viz., plant height (86.3 cm), number of tillers
per hill (12.3), number of leaves per hill (58.6) and total dry matter production (29.45 g
hill-1).
There was a significant increase in the activity of dehydrogenase and phosphatase
enzymes up to 60th DAT in the chlorpyrifos treated soils with all the three efficient
chlorpyrifos degrading inoculants.
A similar trend was observed in the population of microflora in the soils. It was
significantly higher in T9 that received combined treatment of inoculants which might be
due to the synergetic effect of the isolates.
The single inoculation in T4 (CP + CDB-11) recorded a higher degradation of 98.46

% with a residual concentration of 6.325 mg kg-1 followed by the combination of all the
three inoculations (T9) with 95.92 % degradation and 16.767 mg kg-1 of residual
concentration. The pot received chlorpyrifos as sole treatment recorded a lower reduction
(50.77 %) and higher residual concentration (202.20 mg kg-1).
Conclusion
The present study reported a high rate of chlorpyrifos degradation by bacterial

strains exhibiting various growth promoting activities isolated from the rhizosphere of rice.
A total of 25 bacterial isolates were isolated from 40 soil samples collected from different
rice growing areas. It was observed that various species of Pseudomonas and Bacillus
possess a good potential to biodegrade chlorpyrifos effectively. In the midst of
biomagnification of pesticides and contamination of agricultural lands with indiscriminate
use of pesticides, escalating the environmental pollution and deterioration of soil health,
developing a bioremediating agent for mass multiplication and commercialization assumes
a greater significance which can bring down the toxicity levels of the pesticides. Therefore,
isolates CDB-6, CDB-11 and CDB-18 selected in the present study would be ideal
bioremediating agents for lowering the levels of chlorpyrifos. The role of these isolates in
improving growth of the rice plants in chlorpyrifos contaminated site proved their
bioremediating potential both under in vitro and in vivo conditions in addition to phyto-
stimulation which helps to save a considerable amount of inputs in sustainable cultivation
of rice grown in the soils suffering from the toxicity of chlorpyrifos.
Future line of work
1. Mechanisms of chlorpyrifos degradation
2. Isolation of the particular gene/enzyme concerned with the degradation
3. Gene manipulation for the higher degradation capacity
4. Compatibility of the isolated strains with other biofertilizers
5. Multilocation trials for the in vivo evaluation
6. Molecular characterization of the isolates

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Appendix
APPENDIX-I
I. Composition of different growth media/reagents/indicators used
1. MSM broth (Minimal salt media)
Chlorpyrifos 50µl
KH2PO4 4.8g
K2HPO4 1.2g
NH4NO3 1.0g
MgSO4.7H2O 0.2g
Ca (NO3)2.4H2O 0.04g
Fe (SO4)3 0.001g
pH 7.0
2. MR – VP broth
Buffered peptone 7.00 g

Dextrose 5.00 g
Dipotassium hydrogen phosphate 5.00 g
Distilled water 1000 ml
pH 6.9 ± 0.2
3. Nutrient Agar
Peptone 5.00 g
Beef extract 3.00 g
Sodium chloride 5.00 g
Agar 18.00 g
pH 6.8 – 7.2
4. Pikovskaya’s Agar
Yeast extract 0.50 g

Dextrose 10.00 g
Calcium phosphate 5.00 g
Ammonium sulphate 0.50 g
Potassium chloride 0.20 g
Manganese sulphate 0.0001 g
Magnesium Sulphate 0.10 g
Ferrous sulphate 0.0001 g
Agar 20.00 g
pH 7.
5. Simmons Citrate Agar
Magnesium Sulphate 0.20 g

Ammonium dihydrogen phosphate 1.00 g
Dipotassium phosphate 1.00 g
Sodium citrate 2.00 g
Bromothymol blue 0.08 g
Agar 18.00 g
pH 6.8 ± 0.1
6. Starch Casein Agar
Starch 10.00 g
Casein powder 1.00 g
Agar 18.00 g
pH 7.2 ± 0.2
7. Nitrate Broth
Peptone 5.0 g
Beef extract 3.0 g
Potassium nitrate 5.0 g
Water 1000 ml
pH 6.8-7.0
8. Gram Stain Solutions
a) Crystal violet solution
Crystal violet 10.0 g

Ammonium oxalate 4.0 g
Ethanol 100 ml
b) Gram’s Iodine solution
Iodine 1.0 g
Potassium iodide 2.0 g
Ethanol 25 ml
c) Counter stain
2.5% safranin in ethanol 10 ml

d) Ethyl alcohol (Decolorizer)
Ethanol 95 ml
9. Salkowski Reagent
35% perchloric acid 50 ml

0.5M FeCl3 1 ml
10. Milk agar
Skim milk powder 10.0 g

Peptone 5.0 g
pH 7.2
Agar 18.0 g
11. Urea broth
Urea broth concentrate 10.0 ml

Sterile distilled water 90.0 ml
Aseptically the urea broth concentrate was mixed with the sterile distilled water
and dispenced into sterile test tubes.
12. SIM agar
Peptone 30.0 g
Beef extract 3.0 g
Ferrous ammonium sulphate 0.20 g
Sodium thiosulphate 0.025 g
pH 7.3
Agar 18.0 g
13. Martins Rose Bengal Agar (MRBA)
Dextrose 10.0 g
Peptone 5.0 g
Potassium di-hydrogen phosphate 1.0 g
Magnesium sulphate 0.5 g
Rose Bengal 0.05 g
Agar 20.0 g
pH 7.2
14. Kusters Agar (KA)
Glycerol 5 ml
Casein 0.30 g
Potassium nitrate 2.0 g
Magnesium sulphate 0.50 g
Potassium di-hydrogen phosphate 2.0 g
Ferrous sulphate 0.01 g
Calcium carbonate 0.2 g
Agar 20.0 g
pH 7.4

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A STUDY ON CHLORPYRIFOS DEGRADING BACTERIA

UNDER RICE (Oryza sativa L.) ECOSYSTEM

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY

Thesis submitted to the

Master of Science (Agriculture)

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY

This is to certify that the thesis entitled “A STUDY ON CHLORPYRIFOS

Place: RAICHUR __________________________

I have immense pleasure in expressing my whole-hearted sense of gratitude and

I take this opportunity to express sincere heartily thanks to my Advisory Committee

I avail this opportunity to express my sincere gratitude to my dearest and beloved

LIST OF FIGURES vii

LIST OF PLATES viii

II REVIEW OF LITERATURE 5-23

Isolation and screening the efficient chlorpyrifos degrading

Characterization of the efficient chlorpyrifos degrading

In vitro screening of the bacterial isolates for their beneficial

Biodegradation analysis of the chlorpyrifos degrading

Evaluation of the effect of efficient pesticide degrading

III MATERIAL AND METHODS 24-40

Collection of soil samples for the isolation of chlorpyrifos

3.2 Characterization of soil samples for chemical properties 24

Isolation and purification of chlorpyrifos degrading bacteria

In vitro screening for the growth of chlorpyrifos degrading

Estimation of per cent chlorpyrifos degradation by efficient

3.6 Characterization of the efficient chlorpyrifos degrading 27

3.7 In vitro screening of the efficient chlorpyrifos degrading 31

3.8 Evaluation of efficient chlorpyrifos degrading bacterial 32

3.8.1 Preparation of inoculants 34

3.4.2 Planting pattern 34

3.4.3 Plant growth parameters 34

3.4.4 Determination of microbial population in the soil 35

3.4.5 Determination of enzymatic activity in the soil 36

Analysis of chlorpyrifos degradation in soil by the efficient

IV EXPERIMENTAL RESULTS 41-87

Collection of soil samples for the isolation of chlorpyrifos

4.2 Characterization of soil samples for chemical properties 41

Isolation and purification of chlorpyrifos degrading bacteria

In vitro screening for growth of chlorpyrifos degrading

Estimation of percent chlorpyrifos degradation by efficient

Characterization of efficient chlorpyrifos degrading bacterial

In vitro screening of the efficient chlorpyrifos degrading

Evaluation of efficient chlorpyrifos degrading bacteria on

4.8.1 Plant growth parameters 66

4.8.2 Soil enzymatic analysis 72

4.8.3 Determination of microbial population in the soil 75

Analysis of chlorpyrifos degradation in soil by the efficient

Collection of soil samples for the isolation of chlorpyrifos

5.2 Physico-chemical analysis of the soil samples 89

Isolation and purification of the chlorpyrifos degrading

In vitro screening for growth of chlorpyrifos degrading

Estimation of percent chlorpyrifos degradation by the

Characterization of the efficient chlorpyrifos degrading

In vitro screening of efficient chlorpyrifos degrading bacteria

Evaluation of efficient chlorpyrifos degrading bacteria on the

VI SUMMARY AND CONCLUSIONS 118-121

VII REFERENCES 122-135

1 Treatment details of the pot culture experiment 33

Dehydrogenase activity of the soil as influenced by the application

Phosphatase activity of the soil as influenced by the application of

Bacterial population of the soil as influenced by the application of

Fungal population of the soil as influenced by the application of

Population of actinomycetes in the soil as influenced by the