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Synthesis and evaluation of guar gum based hydrogels as
carriers of bio agents for Pythium management
By
K.S.V.POORNA CHANDRIKA
DIVISION OF AGRICULTURAL CHEMICALS
INDIAN AGRICULTURAL RESEARCH INSTITUTE
NEW DELHI – 110012
2013
Synthesis and evaluation of guar gum based hydrogels as
carriers of bio agents for Pythium management
By
K.S.V.POORNA CHANDRIKA
A Thesis Submitted to the Faculty of Post-Graduate School,
Indian Agricultural Research Institute, New Delhi
in partial fulfillment of
requirements for the degree of
MASTER OF SCIENCE
IN
AGRICULTURAL CHEMICALS
2013
Approved by:
Advisory Committee Chairperson: ________________________
(Dr. (Mrs.) Anupama)
Members: ________________________
(Dr. Shashi Bala Singh)
_______________________
(Dr. Anil Saxena)
_______________________
(Dr. Pratibha Sharma)
_______________________
(Dr. M. K. Verma)
Dr. (Mrs.) ANUPAMA Division of Agricultural Chemicals
Principal Scientist Indian Agricultural Research Institute
New Delhi -110 012
CERTIFICATE
This is to certify that the thesis entitled “Synthesis and Evaluation of Guar Gum
Based Hydrogels as Carriers of Bio Agents for Pythium Management” submitted to the Faculty
of the Post-Graduate School, Indian Agricultural Research Institute, New Delhi, in partial
fulfilment of Master of Science in Agricultural Chemicals, embodies the results of bona fide
research work carried out by Miss. K. S. V. Poorna Chandrika, under my guidance and
supervision, and that no part of this thesis has been submitted for any other degree or diploma. The
assistance and help availed during the course of investigation as well as source of information have
been duly acknowledged by her.
(Dr. (Mrs.) Anupama)
_________________________ Date: 29th June, 2013
Chairperson, Place: New Delhi,
Advisory committee India
ACKNOWLEDGEMENT
I would like to express my deepest sense of gratitude and indebtedness to Dr. Anupama, principal
scientist, Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi
and my chairperson, advisory committee for her invaluable guidance, constant encouragement,
cooperative attitude, immense patience, useful discussions and peerless criticism during the
course of investigation and preparation of the manuscript. She has been a consistent source of
inspiration, towards the completion of this work.
It is my privilege to express profound sense of gratitude to Dr. Shashi Bala Singh, Co-
chairperson, advisory committee and principal scientist, Division of Agricultural Chemicals for
her constructive and valuable suggestions.
I take pleasure to convey my heartfelt thanks to Dr. Pratibha Sharma, professor, Division of Plant
Pathology, IARI, and member, advisory committee for her invaluable advice and encouragement
endowed during the research work. I implore my feeling of gratitude to Dr. Anil Saxena, Head,
Division of microbiology, IARI for his cordial behaviour and guidance in designing my
experiments. My sincere thanks are to Dr. V. T. Gajbhiye, Head and Dr. Suresh Walia retired
Professor Division of Agricultural Chemicals, Indian Agricultural Research Institute, for
encouraging me and providing facilities throughout this study.
My heartiest thanks are to Mr. Mahesh Paswan, Mr. Yajulu and Mr. Jagadesh for their technical
assistance and untiring help in entire span of my study. My special thanks are to Dr. Jitender
Kumar, Professor, Division Of Agricultural Chemicals and Supradip Saha, Senior scientist for
allowing me to use their lab facilities, Dr. S. C. Dutta, Principal Scientist, Division of Soil
Science and Agricultural Chemistry for his help in X ray diffraction analysis, Dr. Gautam
Chawla, Principal scientist, Division of Nematology for microscope imaging, Dr. V.V.
Ramamurthy, Principal scientist, Division of Entomology for SEM analysis, Dr. Jasvir Singh,
Division of Plant Pathology for TEM and Dr. Ravinder Kaur, Incharge, WTC for providing
pressure plate membrane apparatus facility. I take this opportunity to thank Dr. A.R.Prasad and
Mr. Rajesh Sekhar, Indian Institute of Chemical Technology (IICT) Hyderabad, India for C13
solid state NMR analysis of samples on good will basis. I duly acknowledge Dr. Anil kumar,
Senior Scientist, IASRI and Dr. Abhishek Rathore, Scientist ICRISAT, Hyderabad for their
valuable help in comprehensive statistical analysis of my research data. It gives me pleasure to
specifically mention names of my lab seniors Dr. Dhruba Jyoti Sarkar, Scientist, Division of
Agricultural Chemicals, Pradeep sir, Prithu Sir, Rupesh sir and Miss Priyanka Dhiman whose
constant help and collective efforts during compilation of my thesis made completion of this
venture possible. The unceasing affection and support of seniors Navkishore Sir, Neethu Mam,
Suman Sir, Aparna Mam, Nagamani Mam shall always be in my memory. I extend my heartfelt
thanks to my juniors Supriya, Ujwala, Vikas and Anu kumari. I thank my class mates Shailesh,
Sujan, Ashish, Ali, Somerender for their special concerns.
The endless love, affection, sacrifice and constant inspiration from my parents and my friend
Madhu enabled me to pursue my goals and never admit defeat. From them, I learnt to perform to
the best of my abilities. Lots of regards are reserved for my Ma’am and her husband Dr. Anil
Kumar for their parent like attitude during my anxious moments. Lots of love and thanks to
Paavan and Addamay, children of my chairperson who patiently spared for me their share of her
attention. I thank God for giving me this opportunity and for surrounding me with people who
believe in me.
Finally, the financial assistance provided by the institute in the form of IARI fellowship during
the tenure of research is gratefully acknowledged.
Place: New Delhi K.S. V. Poorna Chandrika
Dated: 29 June, 2013
Dedicated to Anupama Ma’am &
my parents…
CONTENTS
Chapter No. Title Page No.
1 Introduction 1-2
2 Background 3-13
3 Materials and Methods 14-25
4 Research Paper- I 26-48
5 Research Paper- II 49-67
6 Research Paper -III 68-82
7 General Discussion 84-86
8 Summary and Conclusion 87-88
Abstract
Bibliography i-xx
Appendix xxi
LIST OF TABLES
CHAPTER III
Table Title Page No.
1 Description of the compositions used in the bioefficacy study
against Pythium aphanidermatum 24
CHAPTER IV
Table Title Page No.
1 Swelling response of GG-SAP prepared by conventional and
microwave techniques 31
2 Assignment of various peaks in C
13 NMR spectrum of guar
gum 34
3 Effect of monomer to back bone ratio, cross-linker and
initiator concentration on the water absorption of GG-SAP 39
4 Effect of water volume and molar ratio of alkali and monomer
on water absorbency of GG-SAP 40
5 Effect of particle size of back bone on water absorbency on
GG-SAP 41
6 Effect of temperature, water quality and pH on water
absorbency of GG-SAP 42
7 Water absorbency behaviours of GG-SAP in soilless media
and soil at 25⁰ C and 50⁰ C 45
CHAPTER V
Table Title Page No.
1 Assignment of various peaks in C
13 NMR spectrum of guar
gum 55
2 Elemental composition of superporous hydrogels 57
3 Effect of various synthesis parameters of GG-SPH on water
absorbency 65
4 Effect of pH and quality of water on water absorbency of GG-
SPH 66
5 Effect of salt/ fertilizer type and their strength on water
absorbency of GG-SPH 66
6 Effect of cross linker concentration on density and porosity
on SPHs 67
CHAPTER VI
Table Title Page No.
1 Compositions used in the bioefficacy study against Pythium
aphanidermatum 72
2 Viability behaviour of bioagent test compositions on 180
th
day at different storage temperatures 76
3a Shelf life of bioagents individually and in combinations in
formulations with control in 5⁰ C storage temperature 77
3b Shelf life of bioagents in formulations with control at 25⁰ C
storage temperature 78
3c Shelf life of bioagents in formulations with control at 45⁰C
storage temperature 79
4a Bioefficacy of formulations at 5⁰ C storage 80
4b Bioefficacy of formulations at 25⁰ C storage temperature 80
4c Bioefficacy of formulations at 45⁰ C storage temperature 81
LIST OF FIGURES
CHAPTER II
CHAPTER IV
Figure Title Page No.
1 Sporangia of Pythium aphanidermatum 4
2 General scheme synthesis of superporous and superabsorbent
polymers 7
3 Morphology of dried superporous hydrogel (A) &
conventional hydrogel (B) 7
4 Structure of Guar gum 10
5 Symptoms caused by Pythium aphanidermatum 11
Figure Title Page No.
1 FT-IR spectra of guar gum, acrylamide and GG-SAP 32
2 Diagramatic representation of formation of a typical cl-gg-g-
(polyacrylate) hydrogel 35
3 SEM images of guar gum (A and B) (Gong et al., 2011) and
representative GG-SAPs (C-D) 36
4 XRD peaks of acrylamide, guar gum and GG-SAP 37
5 Effect of time period on swelling at 50⁰ C and 25⁰ C 43
6 Effect of salt/ fertilizer type and their strength on water
absorbency 44
7 Effect of gel addition on available water from soil (A) and
soil less medium (B) 46
8
Moisture retention curves of soil medium (A) and soil less
media under different matric tensions (pF) in unamended and
amended condition
47
CHAPTER V
9
Moisture release curves of soil medium (A) and soil less
medium (B) under different matric tensions (pF) in
unamended and gel amended condition
48
Figure Title Page No.
1 General steps involved in SPH synthesis 51
2 FT-IR spectra of guar gum, acrylamide and GG-SPH 53
3 C
13 NMR spectrum of monomer (A), cross linker (B) and GG-
SPH (C) 54
4 SEM pictures of nonporous GG-SAP (A) and porous GG-
SPHs (B-D) 56
5 Typical harmonized gelation and foaming processes in the
synthesis of superporous hydrogels 57
6 Effect of method of preparation on water absorbency of GG-
SPH 58
7 Scheme of reaction between foaming aid and sodium
bicarbonate (SBC) 58
8 Effect of foam stabiliser type on water absorbency of GG-SPH 59
9 Effect of foaming aid type on water absorbency of GG-SPH 60
10 Effect of backbone to monomer ratio on water absorbency 61
11 Effect of duration of reaction on water absorbency of GG-SPH 62
12 Effect of time period on water absorbency of GG-SPH vs. GG-
SAP 63
13 Effect of temperature on water absorbency of GG-SPH 63
CHAPTER VI
Figure Title Page No.
1
SEM Images of SAP (A) and SPH (B) carriers; SAP
bioformulations of Thz (C and D); SPH bioformulation of Thz
(E); SAP bioformulation of Pflo (F); compound microscopic
images of Pflo(G) and Thz (H).
74
2
Relative reduction (%) in viability of bioagents in test
formulations (A-C) vis-à-vis controls (D) at different storage
temperatures
82
Appendix
Content Title Page No.
1
Tentative patent application (Abstract) Novel
biopolymeric hydrogel carriers and the process of making
novel bioformulation based thereupon with enhanced shelflife
characteristics
xxi
1
INTRODUCTION
Enhancement of crop productivity and quality is of paramount importance in modern
agriculture. Crop protection is an integral component of modern agriculture and pesticides
comprise a major agro-input in this context. In present era of commercial and high value
agriculture, horticultural crops are front runners for betterment of small and marginal farmers.
India is the second largest producer of fruits and vegetables after China. Unfortunately
productivity of crops suffers due to many fungal and viral diseases. Amongst the fungal
diseases, the most serious one is color or root rot. The disease is caused by Pythium
aphanidermatum and Phytopthora parasitica. Pythium aphanidermatum is a cosmopolitan
pathogen with a wide host range, an aggressive species of Pythium, causing damping off, root
and stem rots, and blights of grasses and fruits. Various synthetic fungicides such as
Metalaxyl-M, Propamocab, etc. are recommended for its control. An efficient management
however requires repeated application of the fungicides leading to serious environmental
concerns. Bio control approaches such as use of Trichoderma species, Pseudomonas species
and Vesicular arbuscular mycorrhizae has also been extensively reported. However real
potential remains unidentified primarily due to bottlenecks of high temperature and limited
moisture in the soil (Rini et al., 2007).
Water insoluble hydrophilic polymers, commonly called as superabsorbent hydrogels
possess versatile network properties which provide favourable environment to the microbes
and serve as controlled release systems for regulated release of the agrochemicals (Mondal,
2012). In order to reduce the load of acrylic polymers of petrochemical origin, more emphasis
is being given nowadays to introduce biopolymer backbones or inorganic fillers like clay
minerals (Wang et al., 2007; Grestl et al., 1998)
Guar gum (GG) derived from the seeds of guar plant Cyamoposis tetragonolobus
(Leguminosae) is a natural nonionic branched polymer with β-D-mannopyranosyl units
linked (1–4) with single membered α-D-galactopyranosyl units occurring as side branches.
Native GG and its derivatives have been used in many fields (e.g. thickening agent, ion
exchange resin and dispersing agent, etc.). Guar gum is a low cost, easily available, non-toxic
and biodegradable polysaccharide. Its potential in hydrogel chemistry and new generation
formulation technology still lay less exploited as compared to other backbones like starch,
cellulose, chitosan, xanthan gum etc.
2
Carrier based bioformulation approaches to immobilize and entrap bioagents is catching
worldwide attention (Cho and Lee, 1999). In this context, superabsorbent materials comprise
new generation carriers with tailor made matrix properties.
The present work is thus envisaged with the following objectives:
1. To synthesize and characterize guar gum (GG) based superabsorbent polymers (SAP) and
super porous hydrogels (SPH)
2. To entrap microbial bioagents (Trichoderma harzianum and Pseudomonas fluorescens)
effective against Pythium aphanidermatum, individually or in combinations and
standardize w.r.t shelf life characteristics
3. To evaluate the efficacy of the formulation(s) so prepared against Pythium
aphanidermatum in vitro
3
BACKGROUND
2.1 General
Agriculture continues to play a major role in Indian economy, contributing approximately
12.1% of the total national gross domestic production (GDP) (Economic survey of India
2011-12). In modern agriculture, management of water, nutrients and crop protection inputs
of chemical and biological origin has become higher relevant particularly in view of the fast
changing climate, shrinking natural resources and pest resistance. In Indian agriculture,
horticultural crops play a major role. Some horticultural crops have commercial importance
because of its high nutritive and medicinal value. India leads the world in production of fruits
and vegetables. Unfortunately many crops suffer from many fungal and viral diseases.
Amongst the fungal diseases, the most serious one is color or root rot disease. It is caused by
Pythium aphanidermatum and Phytopthora parasitica.
Pythium aphanidermatum (Edson) Fitz. belongs to a genus of oomycetes pathogens
that cause serious seedling diseases (seedling blight and damping-off) of nearly all field and
horticultural crops worldwide (Agrios, 1988; Martin 1990, 1992; Martin and Loper, 1999).
Poorly drained soil in either fields or greenhouse containers is conducive to seedling infection
by this organism. Chen et al. in 1996 reported that P. aphanidermatum (Martin and Hancock,
1987) infected cucumber seedling by a synergistic action of polygalacturonase (PG) with
other cell wall degrading enzymes. Pectinase was induced when the fungus was grown on
healthy hypocotyls tissue or more effective, on their cell wall. In this action healthy plants
could secrete the exudates contained some cell wall degrading enzymes but their production
and activities were lower than those from the pathogens. P. aphanidermatum (Martin and
Hancock, 1987) colonize seedlings shortly after germination. Production of lytic enzymes has
been proposed to contribute significantly to the virulence of P. aphanidermatum on plant
hosts, but proof of the involvement of these enzymes in disease induction is lacking (Martin
1995). The disease is characterized by the appearance of water-soaked patches on the stem
near the ground level. These patches enlarge rapidly and girdle the stem, causing rotting of
the tissues, which then turn dark brown or black. Such affected plants withstand strong wind,
topple over and die. If the disease attack is mild, only one side of the stem rots and the plants
remains stunted. Fruits if formed, are shrivelled and malformed and gradually the plant dies.
4
Figure 1: Sporangia of Pythium aphanidermatum
Pythium diseases are controlled primarily by cultural and chemical practices. Among
chemical strategies synthetic fungicides such as Metalaxyl-M, Propamocab, furfuryl etc. are
recommended. These fungicides offer temporary solution and require 2-3 applications for a
single crop. The development of fungicide resistance in the pathogens is a major cause of
concern (Singh et al., 1995). Biological control approaches involving use of nonpathogenic
antagonistic microorganisms, especially Trichoderma, Pseudomonas, Bacillus, spp., are
being viewed nowadays an interesting alternative disease control measure (Verma et al.,
2007). The mechanisms for the biocontrol of plant pathogens include competition,
mycoparasitism, antibiosis and induced resistance of the plant host (Chet, 1987; Schirmbock
et al., 1994).
Trichoderma species have been exhaustively explored, formulated and established as
highly efficient bio-control option. Enzymes such as chitinases and/or glucanases produced
by the biocontrol agent are responsible for suppression of the plant pathogen. (Howell, 2003).
Pseudomonas spp. belongs to a group of rhizospheric bacteria described as biological control
agents. These bacteria show great promise with respect to protecting plant roots by reducing
the incidence of fungal-induced diseases (Weller, 1988). Pseudomonads produce versatile
catabolic and secondary metabolites which include antifungal compounds. They have
excellent root-colonizing abilities allowing them to be effective in the vicinity of plant roots.
The soil-borne fluorescent pseudomonads have received special attention and emerged as the
largest and potentially most promising group involved in biocontrol of plant diseases
(DeLaFunte et al., 2004). The bacterium acts by i) producing antibiotics such as 2,4-
5
diacetylphloroglucinol (DAPG) and other phenazine derivatives and ii) through competition
for iron through production of siderophores to kill pathogen (Salman, 2010).
In order to harness the biocontrol potential of Trichoderma and Pseudomonas species,
various efforts have been made worldwide to formulate the bio-agent conidia and/ hyphae
into inert carriers or liquid form (Taweil et al., 2010; Mandhare and Suryavanshi, 2005;
Dubey et al., 2009; Jeyarajan et al., 1994). Temperature and moisture content play
determinant role in the shelf-life and viability of the bioformulations (Rini et al., 2007).
One of the recent technologies for the formulation of biocontrol organisms is the
immobilization of wet or dry biomass within cross linked polymers such as alginate and
carrageenan (Cho and Lee, 1999). Walker and Connick (1983) reported wet and dry
immobilization of biocontrol agents as formulated pellets. Kucuk and Kivanc (2005)
developed zeolite based dry formulation of Trichoderma harzianum which exhibited more
viability of conidia at 40⁰C as compared to 30⁰C. Dubey et al., (2009) reported formulations
of Trichoderma species for soil application wherein sodium alginate, aluminium silicate and
sabudana powder were used for entrapment of bio-agent conidia. Wettable powder and
granule formulations of Trichoderma (Kaewchai et al., 2009; Montealegre et al., 2010) have
been developed. Nakkeeran et al., (2005) remarked that for P. fluorescens’s field application
development of commercial formulations with suitable carriers that support survival of the
bacteria for a considerable length of time is necessary. P. fluorescens, carboxymethyl
cellulose and mannitol showed best shelf life as it maintained highest population recovery at
different DAS (Bora and Deka, 2007). Populations of fluorescent pseudomonads did not
decrease in the talc mixture with 20% xanthan gum after storage for 2 months at 4°C
(Kloepper and Schroth, 1981), and in the vermiculite-based dried formulation after storage
for 6 months at 4°C (Connick, 1988).
New generation carrier approaches in the present context is use of a special class of
polymers called hydrogels. Hydrogels are of two types based on porosity i.e., superabsorbent
polymers (SAP) and superporous hydrogels (SPH). Superabsorbent hydrogels are new
generation carriers finding extensive application in tissue engineering, dewatering sludges,
mining separations, food processing, personal hygienic products, agriculture and other
specialized areas like controlled delivery system (Buchholz and Peppas, 1994).
The structure of the superabsorbent polymer affects its performance in specific use
situations such as agriculture. These polymers are physically or chemically cross linked and
6
can absorb large amounts of water many times its own weight, without dissolving in water
and retain their shape without affecting mechanical properties. Absorbed water is then
released slowly into the surroundings through the evaporation process. This feature has
enabled the use of SAPs in agriculture as water retention aid in soil (Singh et al., 2010,
Zohuriaan-Mehr and Kabiri, 2008)
The actual use of hydrogel in agricultural sector was visualised in 1950s by United
States Department of Agriculture (USDA). This led to development of water soluble
polymers such as polyvinyl acetate (PVA), polyethylene glycol (PEG) and polyacrylamide
(PAM) to function as soil conditioners followed by the introduction of water-swellable
polymeric hydrogels in the early 1980s (Zohuriaan-Mehr and Kabiri, 2008 ). Water-swellable
hydrogels from cross linked polyacrylamide, polyacrylates and copolymers of acrylamide and
acrylates for such applications have been reported extensively (Davies and Castro-Jimenez,
1989; Wang and Boogher 1989; Bres and Weston, 1993; Fonteno and Bilderback, 1993).
Since last three decades; these materials have been tried as soil additives in different
agricultural situations such as dryland agriculture, horticulture, nursery raising, floriculture,
land scaping etc (Singh et al., 2010, Zohuriaan-Mehr and Kabiri, 2008)
In early 1990s, superporous hydrogels (SPHs) were introduced as another category of
water-absorbent polymer systems (Omidian and Park, 2002). Superporous hydrogels are
hydrogels that, regardless of their size, swell to their equilibrium in aqueous media in shorter
duration than the superabsorbent hydrogels. Intially, superporous hydrogels were developed
as gastric retention devices due to fast swelling irrespective of size, large surface area and fast
mass transfer (Chen et al., 1998).
The most important difference between SAP and SPH is their swelling characteristics
mainly related to elasticity of the network, the presence of hydrophilic functional groups
(such as -OH, -COOH, -CONH2, -SO3H) in the polymer chains, the extent of cross-linking,
and porosity of the polymer. Additionally, the physical characteristics of hydrogels including
their swelling ratio also depend on the balance between attractive and repulsive ionic
interactions and solvent mediated effects (Barbieri et al., 1998; Bhalerao et al., 1998).Due to
the presence of interconnected pores in SPH matrix, water can be rapidly absorbed by
capillary attraction forces within the pores, and these polymers swell to their maximum
volume very quickly (Chen et al., 1999; Lee et al., 1997). These hydrogels have the ability to
sense changes of pH and temperature, or the concentration of metabolite and accordingly,
they release their load. SPHs are generally prepared by copolymerization/ crosslinking of co-
7
monomers or crosslinking of linear polymers by irradiation or by chemical compounds
(Satish et al., 2006). Gas blowing techniques are also used to synthesize SPHs (Omidian et
al., 2005). Generalised scheme of synthesis of SAPs and SPHs asdescribed by Omidian et al,
(2005) is shown in Figure 2.
Polymeric materials often have been prepared as carriers for sustained release (Kost et
al., 2001).
Figure 2: General scheme of superporous and superabsorbent polymers (Omidian et al.,
2005)
Figure 3: Morphology of dried superporous hydrogel (A) & conventional hydrogel (B)
(Omidian et al., 2005)
Superporous hydrogels have been explored to develop controlled release
formulations of soil applied pesticides and nutrients / fertilizers and other plant growth
additives. Polyelectrolytic superabsorbents and superporous hydrogels exhibit swelling
response to the external pH and ionic strength, which serves as the switch for controlled
release. Controlled release formulations of various insecticides, herbicides, pheromones and
8
other bioactive molecules entrapped in the superabsorbent and superporous hyrogel
microbeads have been developed through inverse phase suspension polymerization (Chavda
and Patel, 2010). Biopolymeric microspheres of sodium alginate and starch using CaCl2 as a
cross linker are used as carriers for the controlled release of the pesticides, nutrients and
microbes (Roy et al., 2009).
Matrix properties of hydrogel have been utilized to develop an entomopathogenic
nematode Steinernema thermophilum based biopesticidal formulation in our lab (Ganguly et
al., 2008) and a plant growth promoting rhizobacteria based formulation with improved shelf
life characteristics. Integrated formulations of root extracts of Tagetes sp. & magnesium
sulphate adsorbed jointly onto the hydrogel have been reported (Adaka, 2011) that exhibited
significant nematode control coupled with improvement in yield and fruit quality due to
regulated magnesium availability in rhizosphere. Another formulation developed with bio
agents entrapped in zincated hydrogels in our laboratory reveals a promising shelf life and in
vitro bioefficacy against R.solani (Mondal, 2012).
Encouraged by these findings, the present work was undertaken to develop hydrogel
based novel strategies for the management of Pythium.
The following objectives were envisaged:
1. To synthesize and characterize guar gum based superabsorbent polymers (SAP) and super
porous hydrogels (SPH) as carriers.
2. To entrap microbial bioagents (Trichoderma harzianum and Pseudomonas fluorescens)
effective against Pythium aphanidermatum, individually or in combinations and
standardize w.r.t shelf life characteristics.
3. To evaluate the efficacy of the formulation(s) so prepared against Pythium
aphanidermatum in vitro.
This dissertation embodies results of the investigations carried out to achieve the objectives.
2.2 Research Area I (Objective I):
Synthesis and characterization of guar gum based superabsorbent polymers (SAP) and
super porous hydrogels (SPH)
9
In spite of tremendous potential due to versatile matrix and fluid absorption properties, large
scale use of superabsorbent polymers and superporous hydrogels in agriculture and allied
areas has been limited mainly due to high cost, environmental biodegradation concerns, high
rates of application etc. (Singh et al., 2010). Most of the commercial hydrogels available for
use in agriculture are purely synthetic. derived from nonrenewable petroleum resources
(essentially acrylics) due to their superior price to efficiency balance (Davies and Castro-
Jimenez, 1989). The SAPs and SPHs used in agriculture and pharmaceuticals are
polyelectrolyte gels, often composed of acrylamide (AM), acrylic acid (AA) and potassium
acrylate. Therefore, they swell much less in the presence of monovalent salts and collapse in
the presence of multivalent ions (Durmaz and Okay, 2000). These ions can be naturally
provided by soil or introduced through the application of fertilizers and pesticides. Of late, in
view of the environmental concerns all over the world, the importance of replacing the
synthetics by “greener” options is being felt (Zohuriaan-Mehr and Kabiri, 2008). One of the
most popular approaches explored for the purpose is introduction of polysaccharides/
carbohydrates through a suitable mechanism. Chitin, cellulose, starch, and natural gums (such
as xanthan, guar and alginates) are finding extensive application because of their low cost,
natural abundance and availability (Zohuriaan-Mehr, 2006).
Unlike the hydrogels employed in hygiene applications where fast fluid absorption
and retention are intended, for agricultural purpose hydrogels should have the ability to
release the water gradually as per the requirements of plants.
Biopolymers based hydrogels are of current interest due to the environmental
concerns for purely synthetics. Due to their natural origin and of biodegradable character they
are likely to have lesser negative impact on environment (Peterson and Oksman, 2006). A
number of natural polymers such as starch, cellulose, chitosan, pectin, alginate etc. have been
extensively used to develop superabsorbents with versatile applications (Castle et al., 1990;
Kurita, 2001; Lee et al., 1999).
10
Figure 4: Structure of Guar gum
Of late, in view of the inherent hydrophilic characteristics of guar gum (GG) coupled
with their low cost and environment friendly structural properties, guar gum based hydrogels
are gaining prominence in worldwide research on superabsorbent polymers.(Peterson and
Oksman, 2006) Guar gum (GG) is a polysaccharide originating from the seed endosperm of
the plant Cyamopsis tetragonolobus. It is a galactomannan, which consists of a (1→4) linked
β-mannopyranosyl backbone partially substituted at O-6 with α-d-galactopyranosyl side
groups, with the ratio of mannose to galactose ∼1.6–1.8:1(Cheng et al., 2002). It has been
extensively used in a number of applications such as thickening agents, suspending agents,
ion-exchange resins, surfactant and dispersing agent,). Owing to the hydrophilic properties of
guar gum due to the presence of hydroxyl groups, preparation of guar gum based
superabsorbent polymers (SAP) and superporous hydrogels (SPH) is being considered a
promising approach (Peterson and Oksman, 2006; Chen et al., 2000; Kim and Park, 2004).
Guar gum has been employed in the present work to develop and evaluate crosslinked
superabsorbent and superporous hydrogels with varying fluid absorption and matrix
characteristics with an aim to develop potential carriers of microbes.
11
2.3 Research Area II (Objective II):
Entrapment of microbial bioagents (Trichoderma harzianum and Pseudomonas
fluorescens) effective against Pythium aphanidermatum, individually or in combinations
and standardize w.r.t shelf life characteristics
In present era of commercial and high value agriculture, horticultural crops are front runners
for betterment of small and marginal farmers. India leads the world in production of fruits
and vegetables. Unfortunately many crops suffer from many fungal and viral diseases.
Amongst the fungal diseases, the most serious one is color or root rot disease. (Kannan &
Jayaraj, 1998) It is caused by Pythium aphanidermatum and Phytopthora parasitica etc.
Pythium aphanidermatum is a severe pathogen which is responsible for many diseases in
several crops in young and mature ages.
The disease is characterized by the appearance of water-soaked patches on the stem
near the ground level. These patches enlarge rapidly and girdle the stem, causing rotting of
the tissues, which then turn dark brown or black (Figure 5). Such affected plants withstand
strong wind and topple over and die. If the disease attack is mild, only one side of the stem
rots and the plants remains stunted. Fruits if formed are shrivelled and malformed. Gradually
the plant dies.
Figure 5: Symptoms caused by Pythium aphanidermatum
Pythium spp. are worldwide in distribution (Hendrix and Campbell, 1973) that attack
cuttings, seeds, seedlings and all stages of the various crops causing significant losses to
them. Almost all greenhouse crops are susceptible to one or more species of Pythium (Miller
and Sauve, 1975; Stephens and Powell, 1982). Of the different species of Pythium, P.
aphanidermatum (Edson) Fitz. is reported from a large number of hosts (Van der Plouts-
Niterink, 1981).
12
The most common approach to check the diseases caused by P. aphanidermatum in
plants is use of chemical fungicide (Ulrike and Werner, 1998; Salman and Abuamsha, 2012).
The increasing awareness of fungicide-related hazards has emphasized the need of adopting
biological methods as an alternative disease control approach. Species of the genus
Trichoderma are well documented fungal biocontrol agents (Papavizas, 1985; Elad and
Kapat, 1999; Howell, 2002). The antagonistic action of Trichoderma species against
phytopathogenic fungi might be due to either by the secretion of extracellular hydrolytic
enzymes (Chet, 1987; Di Pietro et al.,., 1993; Schirmbock et al., 1994) or by the production
of antibiotics (Dennis and Webster, 1971a; Dennis and Webster, 1971b; Claydon et al., 1987;
Howell, 1998). Nonpathogenic bacteria of Pseudomonas spp. are also effective root
colonizers and biocontrol agents antogonising by production of antibiotics and other
antifungal metabolites, hydrogen cyanide and siderophores (O'Sullivan and O'Gara, 1992).
In recent years, more emphasis is laid on the combined use of biocontrol agents with
different mechanisms of disease control, for improved disease control and also to overcome
the inconsistent performance of the introduced biocontrol agent. (Chet, 1987; Howell and
Pukhaber, 2005; Lewis and Papavizas, 1985)
The present work aims at a) employing the guar gum based hydrogels developed in
objective to develop composite formulations of Trichoderma harzianum spores/ mycelia and
Pseudomonas fluorescens spores entrapped together and individually evaluate their shelf life
characteristics over a range of storage temperatures as applicable.
2.4 Research area (Objective III):
Bioefficacy evalution of the formulation(s) so prepared against Pythium
aphanidermatum in vitro
Hydrogels are established to influence the water retention, infiltration capacity of the soils
particularly sandy loam soils (Bhardwaj et al., 2007), behaving typically as organic matter.
Therefore, their use as bioagent carriers is expected to influence the interaction of bioagent
with the target pathogens and enhance the shelf life characteristics due to hydrophilic
behaviour. In India, use of hydrogels in dryland agriculture and hi-tech vegetable cultivation
is finding increasing recommendation as an innovative water management (Anupama, 2005).
In a previous work reported from our laboratory, Trichoderma harzianum-nutrient combo
formulation based on hydrogels has been developed (Mondal and Anupama, 2012) which
13
exhibits significant bioefficacy against Rhizoctonia solani (in vitro). To the best of our
information, no work has been reported on the composite formulations of biocontrol agents
employing hydrogels as carriers. The present work was undertaken to evaluate the
bioefficacy of the integrated hydrogel based formulations of Trichoderma harzianum and
Pseudomonas fluorescens in vitro developed in the objective-II.
14
MATERIALS AND METHODS
Enhancement of crop productivity and quality is of paramount importance in modern
agriculture. Crop protection is an integral component of modern agriculture and pesticides
comprise a major agro-input in this context. In present era of commercial and high value
agriculture, horticultural crops are front runners for betterment of small and marginal farmers.
India is the second largest producer of fruits and vegetables after China. Unfortunately
productivity of crops suffers due to many fungal and viral diseases. Amongst the fungal
diseases, the most serious one is color or root rot. The disease is caused by Pythium
aphanidermatum and Phytopthora parasitica. Pythium aphanidermatum is a cosmopolitan
pathogen with a wide host range, an aggressive species of Pythium, causing damping off, root
and stem rots, and blights of grasses and fruit. Various synthetic fungicides such as
Metalaxyl-M, Propamocab, etc. are recommended for its control. An efficient management
however requires repeated application of the fungicides leading to serious environmental
concerns. Bio control approaches such as use of Trichoderma species, Pseudomonas species
and vesicular arbuscular mycorrhizae has also been extensively reported. However real
potential remains unidentified primarily due to bottlenecks of high temperature and limited
moisture in the soil (Rini et al., 2007).
Water insoluble hydrophilic polymers, commonly called as superabsorbent hydrogels
possess versatile network properties which provide favorable environment to the microbes
and also serve as controlled release systems for regulated release of the agrochemicals
(Mondal, 2012). In order to reduce the load of acrylic polymers of petrochemical origin, more
emphasis is being given nowadays to introduce biopolymer backbones or inorganic fillers
like clay minerals (Wang et al., 2007; Grestl et al., 1998)
Guar gum (GG) derived from the seeds of guar plant, Cyamoposis tetragonolobus
(Leguminosae), is a natural nonionic branched polymer with β-D-mannopyranosyl units
linked (1–4) with single membered α-D-galactopyranosyl units occurring as side branches.
Native GG and its derivatives have been used in many fields (e.g. as thickening agent, ion
exchange resin and dispersing agent, etc.). Guar gum is a low cost, easily available, non-toxic
and biodegradable polysaccharide. Its potential in hydrogel chemistry and new generation
15
formulation technology still lay less exploited as compared to other backbones like starch,
cellulose, chitosan, xanthan gum etc.
Materials used and methods employed to achieve the objectives of the present investigation
are briefly described below:
3.1 Materials
3.1.1 Reagents for synthesis of hydrogels
Guar gum (LR), acrylamide (LR), N,N’- methylene bis acrylamide and a per sulphate
initiator were purchased from Thomas Baker Pvt Ltd., Mumbai, India. N,N,N’,N’-
Tetramethyl ethylene diamine (TEMED) (GR), surfactants FS-1, FS-2, FS-3 and FS-4, GR
(99% assay) and carboxylic acids OA-1, OA-2 and OA-3 were purchased from Merck
Specialities Pvt Ltd., Mumbai, India. Sodium bicarbonate (99.5% minimum assay) was
procured from Sd Fine Chem Pvt Ltd., Mumbai, India.
3.1.2 Plant pathogenic fungus (Pythium aphanidermatum)
Fresh live culture of Pythium aphanidermatum was obtained from ITCC (Indian Type
Culture Collection) section of Plant Pathology Division, Indian Agricultural Research
Institute, New Delhi, India. The culture was maintained on PDA slants in BOD incubator at
28 ± 2°C for multiplication and was sub-cultured in test tubes and Petri dishes prior to
testing.
3.1.3 Biocontrol
Fresh live culture of Trichoderma harzianum fungus was obtained from ITCC (Indian Type
Culture Collection) section of Plant Pathology Division, Indian Agricultural Research
Institute, New Delhi, India. The culture was maintained on Potato dextrose agar (PDA) slants
in BOD incubator at 28 ± 2°C for multiplication and was sub-cultured in test tubes and Petri
dishes prior to testing. Fresh live culture of Pseudomonas fluorescens bacteria was obtained
from ITCC (Indian Type Culture Collection) section of Plant Pathology Division, Indian
Agricultural Research Institute, New Delhi, India. The culture was maintained on Nutrient
agar (NA) slants in BOD incubator at 28 ± 2°C for multiplication and was sub-cultured in test
tubes and Petri dishes prior to testing.
16
3.1.4 Fungal growth media
Analytical grade Potato Dextrose Agar (PDA) media was purchased from Merck Specialities
Pvt. Ltd., Mumbai, India (pH 5.6 ± 0.2 at 250C). Analytical grade Potato Dextrose Broth
(PDB) media was purchased from Hi-media Laboratories Pvt. Ltd., Mumbai, India (pH 5.1 ±
0.2 at 250C).
3.1.13 Bacterial growth media
Analytical grade Nutrient Agar (NA) media was purchased from Merck Specialities Pvt. Ltd.,
Mumbai, India (pH 5.6 ± 0.2 at 250C). Analytical grade Nutrient Broth (NB) media was
purchased from Hi-media Laboratories Pvt. Ltd., Mumbai, India (pH 5.1 ± 0.2 at 250C).
3.2 Methods
3.2.1.1 Synthesis of superabsorbent polymers
In situ solution polymerization was used to synthesize the biopolymeric SAPs taking various
ratios of backbone, vinyl monomer, cross linker and initiator in a definite volume of water at
a particular temperature. The alkali was used either during in situ reaction or after gel
formation. Gel was suitably treated to attain pH of 7.0 and dried to get xerogel. The reaction
parameters were standardized using sequential selection procedure.
3.2.1.2 Backbone: monomer ratio
Mixture of backbone, cross linker and initiator was taken in a particular ratio and reacted with
different concentrations of monomer (weight %). Rest of the procedure adopted was same as
mentioned above. Monomer content that resulted in a SAP exhibiting maximum absorption in
distilled water was taken for the next step.
3.2.1.3 Cross linker concentration
Mixture of backbone, monomer and initiator was taken in a particular ratio in water and
reacted with different concentrations of cross linker (weight %). Rest of the procedure was
same as mentioned above. Cross linker content resulting in a SAP with maximum absorption
in distilled water was taken for the next step.
17
3.2.1.4 Initiator concentration
Mixture of Backbone, monomer and crosslinker was taken in a particular ratio in water and
reacted with different concentrations of initiator (weight %). Rest of the procedure adopted
was same as mentioned above. Initiator content resulting in a SAP with maximum absorption
in distilled water was taken for the next step.
3.2.1.5 Quantity of water per unit reaction mass
The optimized composition obtained from the above variations was reacted in the presence of
various quantities of water (ml/gm) per gram reaction mass. Rest of the procedure adopted
was same as mentioned above. Water volume resulting in SAP with maximum absorption in
distilled water was taken for the next step.
3.2.1.6 Particle size of backbone
Mixture of backbone, monomer, initiator, cross linker was taken in standardized quantity of
water. Particle size was varied from 100 to >240 mesh size.
3.3 Absorption study of hydrogel in salts and different pH solutions
Solutions of different strengths (5mM, 10mM, 15mM and 20mM ) of NH4SO4, NH4NO3,
KNO3, NaCl and Urea each were prepared and used for evaluation of swelling behaviour of
SAP. Buffer solutions of pH 4, 7, 9 were prepared and used for absorption studies.
3.4 Water absorption and retention study of amended soil and soilless media
Sandy loam soil (pH: 7.8 as measured at 1:1.25 soil to water ratio; organic carbon content:
0.51% for natural soil as determined by Walkley and Black method (Jackson, 1967); soil
mechanical fractions: sand 78%, silt 10%, clay 12.4%, employing Bouycos hydrometer
(Black et al., 1965) method; cation exchange capacity: 11.2 Cmol/ kg by normal ammonium
acetate method (Jackson, 1967) (pH 7.0)) and soil less media (a sterlized mixture of cocopeat,
vermiculite and perlite in the ratio 3:1:1 on volume basis) were collected from institute farm
and nursery. Air dried soil sample was passed though the 2 mm sieve and mixed with SAP at
the rate of 0.5% and 0.75% each. For water absorption measurement, amended soil (50 g) or
soilless medium (20 g) was taken in preweighed plastic cups having perforated base fitted
with filter paper. Each cup was immersed overnight in enough water to allow its capillary
18
rise. Water holding capacity (WHC) of soil and soilless medium was calculated by
gravimetric method using following equation:
WHC (%) = M-m/m × 100
Where M denotes weight of wet soil mix and m denotes weight of oven dry soil mix. Water
retention study was done using pressure plate apparatus at different tensions-2.3 pF, 2.8 pF,
3.0 pF, 3.7 pF, 4.0 pF and 4.2 pF. Ceramic plates were kept overnight in water for saturation.
Amended soil and soilless media were filled in rubber rings arranged on bar plates and
allowed to saturate overnight. Care was taken to ensure proper contact between the samples
and ceramic plate surface. The saturated samples along with ceramic plates were placed in
pressure chamber pertaining to different tensions. The pressure was applied and maintained
till water stopped flowing out of the chamber. Samples were transferred to moisture boxes
immediately and weighed. The moist samples were dried in a hot air oven at 105oC for 24
hours, air cooled and reweighed. The amount of water held at particular pressure was
calculated using following equation:
WC (% by weight) = W-w / w × 100
Where WC is the percent water content of soil or soilless media on weight basis, W is the
mass of wet soil or soilless media at a particular tension and w is the weight of oven dried
soil or soilless medium (amended or control).
3.5 Synthesis of superporous hydrogels
In situ solution polymerization was used to synthesize superporous hydrogels taking various
ratios of backbone, vinyl monomer/s, cross linker, initiator, foam stabilizer ratio, porogen in a
definite volume of polar solvent/s at a particular temperature for a definite time period.
Different drying methods were used to obtain dry hydrogels. The reaction parameters were
standardized using sequential procedure as follows-
3.5.1 Foam stabilizer type
Mixture of backbone, cross linker, monomer, initiator and reductant, foaming aid and
porogen was taken in a particular ratio in the feed mixture. Rest of the procedure adopted was
same as mentioned above. The type of foam stabilizer that resulted in a SPH exhibiting
maximum absorption in distilled water, was taken for the next step. Four foam stabilizers
19
belonging to different chemical classes FS-1, FS-2, FS-3 and FS-4 were tried complete
details will be protected under IPR. Rest of the procedure was the same as mentioned above.
3.5.2 Monomer concentration
Mixture of backbone, cross linker, initiator and reductant, foam stabilizer, porogen and
foaming aid was taken in particular ratio of water and reacted with different concentrations of
monomer (weight %) from. Rest of the procedure adopted was same as mentioned above.
Monomer content that resulted in SPH exhibiting maximum absorption in distilled water was
taken for the next step.
3.5.3 Cross linker concentration
Mixture of backbone, monomer, initiator and reductant, foam stabilizers, porogen, and
foaming aid was taken in a particular ratio in water and reacted with different concentrations
of cross linker (weight %) of total feed mixture. Rest of the procedure adopted was same as
mentioned above. Cross linker content that resulted in a SPH exhibiting maximum absorption
in distilled water was taken for the next step.
3.5.4 Foam stabilizer concentration
Mixture of backbone, cross linker, monomer, initiator and reductant, porogen and foaming
aid was taken in a particular ratio in water and reacted with foam stabilizer (selected as ibid).
Rest of the procedure adopted was same as mentioned above. The foam stabilizer content that
resulted in a SPH exhibiting maximum absorption in distilled water was taken for the next
step.
3.5.5 Quantity of water per unit reaction mass
The optimized composition obtained as above was reacted in the presence of varying
quantities of water (mL/g). Rest of the procedure adopted was same as mentioned above.
Water quantity per gram reaction mass that resulted in a SPH exhibiting maximum absorption
in distilled water was taken for the next step.
20
3.5.6 Porogen concentration
Mixture of backbone, cross linker, monomer, initiator and reductant, foam stabilizer, foaming
aid was taken in a particular ratio in water and reacted with different concentrations of
porogen (weight %). Rest of the procedure was followed which was mentioned above. The
concentration of porogen that exhibited maximum water absorption in SPH was taken to next
step.
3.5.7 Foaming aid type
Mixture of backbone, cross linker, monomer, initiator and reductant, foam stabilizer, foaming
aid was taken in a particular ratio in water and reacted with different types of foaming acids
OA-1, OA-2, OA-3 and OA-4. Rest of the procedure was followed as mentioned above. The
type of foaming aid which resulted in maximum water absorption of SPH was taken to next
step.
3.5.8 pH of medium
Mixture of backbone, monomer, cross linker, initiator reductant, foam stabilizer, porogen and
foaming aid was taken in a particular ratio in water. Rest of procedure was followed as
mentioned above. pH of reaction medium was varied from 3.0-6.0.
3.6 Characterization of superabsorbent and superporous hydrogels
Micro Fourier transform infrared spectroscopy (National Physical Laboratory, New Delhi,
India), solid state 13
C nuclear magnetic resonance spectroscopy (BRUKER DSX 300, 7.04
tesla, carbon frequency 75.47 MHz; Indian Institute of Chemical Technology, Hyderabad,
India), elemental analysis (EURO EA elemental analyzer; Indian Agricultural research
Institute, New Delhi, India), scanning electron microscopy (ZEISS EVO Series Scanning
Electron Microscope (EVO 50) with resolution of 2.0 nm @ 30 kV; Indian Agricultural
Research Institute, New Delhi, India), X-ray diffraction technique (PHILIPS PW1710
diffractometer control equipped with PHILIPS PW1728 X-ray generator; Indian Agricultural
research Institute, New Delhi, India) techniques were used to characterize the superabsorbent
and superporous hydrogels synthesized in the present work.
21
3.7 Water absorption study of superabsorbent and superporous hydrogels in distilled
water, tap water and hard water through gravimetric method
The accurately weighed hydrogel (SAP/SPH) (0.1 g, particle size 100-240 mesh size) was
immersed in the excess of distilled water (pH 7.0, EC 0.001 Mhos/cm) in triplicate and kept
at two temperatures, 500 C until equilibrium was attained. The unabsorbed water was filtered
through a nylon sieve (200 mesh size), gel allowed to drain on sieve for 10 minutes and
weighed. The water absorption (Q H2O) was calculated using the following equation:
QH₂O = (w2-w1)/ w1,
Where w1 is the weight of dried absorbent and w2 is the weight of swollen absorbent
at equilibration. QH2O was calculated as grams of water per gram of dry sample. Water
absorption in distilled water was studied as function of time duration (30 min- 24 hours) i.e.,
rate of absorption and temperature (8o, 20
o, 50
o and 70
o C).
3.8 Absorption study of polymer in different salt and pH solution-
Salt solutions of different strengths (5 mM, 10 mM, 15 mM and 20 mM) of NH4SO4,
NH4NO3, KNO3, NaCl and Urea each were prepared and used for evaluation of swelling
behaviour of SPH. Buffer solutions of pH 4, 7 and 9 were prepared and used for absorption
studies.
3.9 Density and porosity measurements
For density measurement, the solvent replacement method was used (Tang et al., 2005). A
piece of dried SPH was taken and weighed. It was immersed in a predetermined volume of
hexane in a graduated cylinder and the increase in hexane in a graduated cylinder and the
increase in hexane volume was measured as the volume of the polymer. The density was
calculated from the following equation:
Density = MSPH/VSPH
Where, VSPH is the volume of solvent displaced by SPH and MSPH is the mass of the
SPH.
22
For porosity measurement, dried hydrogels of different cross linker concentrations
were immersed in hexane overnight and weighed after excess hexane on the surface was
blotted off. The porosity was calculated from the equation:
Porosity = VP/VT
Where, VP = VT - VSPH is the pore volume of SPH and VT is the total volume of the
SPH. Total volume of SPH was measured from its dimensions, as it was cylindrical in shape
(Chavda and Patel, 2009).
3.10 Preparation of bioformulations
3.10.1 In vitro compatibility evaluation of Pseudomonas fluorescens (Pflo) and
Trichoderma harzianum (Thz)
The compatibility between Pseudomonas fluorescens and Trichoderma harzianum was
evaluated in vitro by poisoned food technique using potato dextrose agar (PDA) medium and
nutrient agar (NA) medium (Sahayaraj et al., 2011).
3.10.2 Preparation of media (Marshall, 1993)
For fungi, PDA, 39.5 g was suspended in 1000 mL distilled water. The suspension was boiled
to obtain uniform media. For bacteria, NA, 28 g was suspended in 1000 mL distilled water.
The suspension was boiled to obtain uniform media. For combinations (bacteria & fungi),
mixture of NA and PDA (3:7) was suspended in 1000 mL distilled water. The media (50 ml)
was transferred to conical flasks (100 ml capacity). The flasks were plugged with surgical
grade cotton prior to wrapping with brown paper. The media and Petri dishes were put in
sterilized autoclaved bags and autoclaved at 15 psi for half an hour prior to use. PDB media
was prepared by dissolving 24 g PDB in 1000 mL distilled water. NB media was prepared by
dissolving 13 g NB in 1000 mL distilled water. Same precautions were taken as in case of
PDA media.
3.10.3 Compatibility evaluation of Trichoderma harzianum and Pseudomonas
fluorescens
Sterilized mixture of NA and PDA media (3:7) was poured into 92 mm diameter Petri plates
@ 20 ml per plate and 5 mm diameter solid discs of inoculum of each of the bacterium and
23
fungus was placed near the edges of the Petri plates. Individual inoculum was placed in the
centre of dish separately as control. Three replicates were taken for each treatment. Petri
dishes were incubated in BOD incubator @ 28oC. Growth of each organism was recorded on
5th
day when the upper surface of solidified Petriplate was just full with microbial growth as
in case of control.
Percent compatibility was calculated as: % compatibility = 100 - % inhibition
3.10.4 Bioassay study against Pythium aphanidermatum
In vitro bioassay experiments on Pythium aphanidermatum were carried out by following
poison media method (Sahayaraj et al., 2011).
Following two bioassay experiments were conducted:
a) Antagonistic activity of Trichoderma harzianum and Pseudomonas fluorescens
individually and in combinations
b) Antagonistic activity of hydrogel based combo and individual formulations of
Trichoderma harzianum and Pseudomonas fluorescens.
3.10.4.1 Bioefficacy of Trichoderma harzianum and Pseudomonas fluorescens
individually and in combinations
After solidification of the media poured into the Petri plate, mycelia agar plug of pathogen
was taken from the Petri plate with full growth and placed in the centre. Pathogen was placed
in the centre and biocontrol agent was kept at periphery of Petriplate for individual bioassay.
For joint activity evaluation of Trichoderma harzianum and Pseudomonas fluorescens, the
pathogen was placed at the centre and both bioagents were placed separately on either side of
the Petri plates. Pythium aphanidermatum was used as control.
3.10.6 Bioefficacy of bioformulations
Test composition (0.1 g) from each treatment was uniformly dispersed in the growth medium
in Petri plate. Petri plates were kept at B.O.D. incubator at 280C. After 5-7 days, when the
growth of pathogen became maximum inhibition% was calculated from the reduction of
diameter in treated Petri plates.
24
I = (C – T) / C x 100
Where I = Percent growth inhibition, C = Colony diameter in control and T = Colony
diameter in treatment
Description of various treatments planned for bioefficacy study (in vitro) of the
prepared hydrogel based bioformulations is given in table 1. Bioefficacy of test compositions
stored at 5 0C, 25
0C and 45
0C for a duration of 180 days was evaluated periodically (0, 15,
30, 60, 90, 120, 150 & 180 days).
Table 1: Compositions used in the bioefficacy study against Pythium aphanidermatum
Serial No Treatments
1. Control (Thz)
2. Control (Pflo)
3. Control (Thz and Pflo ;1:1 mixture)
4. Wet formulation of Thz in superabsorbent hydrogel (SAP) (WSAP-Thz)
5. Wet formulation of Pflo in superabsorbent hydrogel (SAP) (WSAP-Pflo)
6. Wet formulation of Thz and Pflo mixture in superabsorbent hydrogel (SAP)
(WSAPC)
7. Dry formulation of Thz in superabsorbent hydrogel (SAP) (DSAP-Thz)
8. Dry formulation of Pflo in superabsorbent hydrogel (SAP) (DSAP-Pflo)
9. Dry formulation of Thz and Pflo in superabsorbent hydrogel (SAP) (DSAPC)
10. Wet formulation of Thz in superporous hydrogel (SPH) (WSPH-Thz)
11. Wet formulation of Pflo in superporous hydrogel (SPH) (WSPHC)
12. Wet formulation of Thz and Pflo in superaporous hydrogel (SPH) (WSPHC)
13. Dry formulation of Thz in superporous hydrogel (SPH) (DSPH-Thz)
14. Dry formulation of Pflo in superporous hydrogel (SPH) (DSPH-Pflo)
15. Dry formulation of Thz and Pflo in superporous hydrogel (SPH) (DSPHC)
25
3.10.7 Preparation of bioformulations of Trichoderma harzianum and Pseudomonas
fluorescens
Novel process of preparation of hydrogel based formulations of Trichoderma harzianum and
Pseudomonas fluorescens was developed in the laboratory. Details of the complete process
will be protected under IPR. Two types of formulations were prepared, dry and wet based on
their physical state of appearance. Precautions were taken to introduce 108-10
11 colony
forming units of bioagents individually or in combination, per gram of carrier. In all, twelve
compositions were prepared (Table 1).
3.11 Shelf-life evaluation to assess viability as a function of time and temperature
All the twelve compositions were kept at three storage temperatures 5 0C, 25
0C and 45
0C in
B.O.D. incubators. Sampling was done periodically at intervals of 0, 15, 30, 60, 90, 120, 150
and 180 days (Mondal, 2012).
3.11 Statistical Analysis.
Statistical analysis of all the laboratory CRD experiments was done using PROC GLM SAS
(9.2) of SAS Institute, Cary, NC.
26
RESEARCH PAPER -I
pH-sensitive cross linked guar gum based superabsorbent hydrogels: synthesis, swelling
response in simulated environments and water retention behaviour in plant growth
media
Abstract
Cross-linked guar gum-g-polyacrylate (cl-gg-g-PA) superabsorbent hydrogels were prepared
to explore their potential as soil conditioners and carriers. The hydrogels were prepared by in-
situ grafting polymerization and cross-linking of acrylamide on to a natural guar gum
followed by hydrolysis. Microwave initiated synthesis under the chosen experimental
conditions did not exhibit any significant improvement over the conventional technique. The
optimization studies of various synthesis parameters, namely, monomer concentration, cross-
linker concentration, initiator concentration, quantity of water per unit reaction mass, particle
size of backbone and concentration of alkali were done. The hydrogels were characterized by
X-ray diffraction, scanning electron microscopy, FT-IR and solid state C13
NMR
spectroscopy. Swelling behaviour of a candidate hydrogel (GG-SAP) in response to external
stimuli namely, salt solutions, fertilizer solutions, temperature, and pH was studied. The GG-
SAP exhibited significant swelling in various environments. Effect of GG-SAP on water
absorption and retention characteristics of sandy loam soil and soil-less medium was also
studied as a function of temperature and moisture tensions. Addition of GG-SAP significantly
improved the moisture characteristics of plant growth media (both soil and soil-less),
showing that it has tremendous potential for diverse applications in moisture stress
agriculture.
Key words: guar gum, swelling, soil conditioner, pH- sensitive
4.1 Introduction
In view of the fast depletion of ground water reserves, uncertainty of rains in arid and
semiarid regions of the world, coupled with the growing food demands of the burgeoning
human population, efficient use of water available for crops has become highly relevant. In
recent years, the use of superabsorbent polymers (SAPs) has been viewed as an innovative
tool to improve water use efficiency in agricultural operations. Since the report of the first
27
SAP by United States Department of Agriculture (Hedrick et al., 1952), the research on
developing better products for use in agriculture continues world-wide. Polymeric soil
conditioners have been known since 1950s (El-Handy et al., 2006).
These polymers were
developed to improve the physical properties of soil such as water-holding capacity (WHC),
soil permeability, and infiltration rates, facilitating plant development, especially in structure
less soils in areas subject to drought (El-Handy et al., 2006, Bhardwaj et al., 2007, Johnson et
al., 1990, Johnson et al., 1984). In addition, purely synthetic as well as semisynthetic
hydrogels have been used for removal of heavy metals from water as well as controlled
release agro-input devices in agriculture (Fonteno et al., 1993). However, the effects of
hydrogel technology in agriculture are inconclusive because contradictory
effects on physical
properties of growing media have been reported (Taylor et al., 1986, Wang et al., 1990, Elliot
et al., 1992, Blodgett et al., 1993, Fonteno et al., 1993, Davies et al., 1989, Wang et al.,
1989, Bres et al., 1993, Martinz et al., 2000, Ahmad et al., 1994, Wallace et al., 1988,
Henderson et al., 1985, Wang et al., 1987). Although a number of commercial hydrogels
have been tried and recommended in agriculture and horticulture, their use on large scale
remains limited primarily because of high cost & rate of application, and their inability
to
perform under harsh agro-climatic conditions (Wang et al., 1990, Falatah et al., 1996). A
SAP of semi synthetic origin ‘Pusa gel’ has already been developed and commercialized
from our laboratory (Anupama et al., 2005). Its potential as a water retentive aid in
agriculture is well established. Most of the soil conditioners reported so far, though possess
impressive fluid absorption characteristics, a candidate SAP for use under alkaline soil
conditions is still elusive. The SAP studied in this work was developed to explore its potential
as an alternative with superior characteristics.
Biopolymeric superabsorbent hydrogels have been receiving great attention because
of the enhanced properties over purely synthetic hydrogels (Falatah et al., 1995; William et
al., 2005; Park et al., 2003, Hule et al., 2007; Singh et al., 2011). Inherent traits of the
biopolymers derived from plant sources can be manipulated through chemical modifications
to achieve desired characteristics in the finished products. Guar gum is one such biopolymer
of interest in SAP chemistry that has been exploited in various areas e.g., thickening agent,
ion exchange, suspending agent, controlled release devices for biomedical applications
(Wang et al., 2010). Guar gum, a non-ionic galactomannan polysaccharide seed gum derived
from Cymaposis tetragonolobus, has also been used in discrete studies to develop SAPs and
28
SAP composites with pH-sensitive characteristics (Zhang et al., 2006; Pourjavadi and
Mahdavinia, 2006; Sadeghi and Hosseinzadeh, 2008). In agriculture, a balance of
water/nutrient retention-release characteristics favourable to plant and ability to perform
under extremes of environmental conditions such as temperature, pH, salinity in soil etc. is an
area of interest. In the present study, crosslinked-guar gum-graft-(polyacrylate) hydrogels
have been synthesised by free radical solution polymerization. The effect of various synthesis
parameters on their water absorbency has been reported and supported by structural
characterization. In the second part of this study, an exhaustive evaluation of their
performance as a function of external stimuli viz. temperature, pH, salts and fertilizers as well
as water absorption, retention and release characteristics in sandy loam, soil and soil less
media has been presented.
4.2 Materials and methods
4.2.1 Chemicals
Commercial Guar gum, acrylamide, N, N- methylenebisacrylamide cross-linker, and a
persulfate initiator were purchased locally from Thomas baker (Pvt) Ltd. Mumbai, India and
used as such without further purification.
4.2.2 Preparation of guar gum-g- polyacrylate hydrogels (GG-g-PA)
The process of synthesis of biopolymeric superabsorbent materials for agricultural
applications has been standardised previously in our laboratory (Anupama et al., 2005) and
the same was used here with minor modifications. Briefly, SAPs were prepared by in situ
grafting polymerization and cross linking of acrylamide on the guar gum backbone in the
presence of cross-linker using persulfate initiator. To optimize the concentration of various
synthesis parameters, a sequential completely randomized design was adopted. A typical
procedure used was as follows: an aqueous solution containing monomer, cross-linker, guar
gum followed by addition of initiator was heated up to 80⁰C for a predetermined period. The
mixture was kept as such for a couple of hours. The resulting product was treated with
different molar ratios of alkali for a fixed period under ambient conditions (Anupama et al.,
2005), washed with distilled water and either air dried or with the help of methanol. The
dehydrated sample was further vacuum dried till constant weight. The dried product was
milled and screened through 100-240 mesh-size screen. FT-IR spectra of native guar gum and
29
an optimized GG-SAP were recorded in KBr discs on a Bruker Fourier Transform Infrared
Spectrophotometer under dry air at room temperature. Solid state C13
NMR spectra of
optimized GG-SAP were recorded on a BRUKER DSX 300, spectroscopy operating at 7.04
tesla, carbon frequency 75.47 MHz for carbon. Cross linker and monomer were characterized
by liquid state C13
NMR using BRUKER Avance 400 instrument. Wide-angle X-ray
diffraction (XRD) measurements were performed using a Philips PW1710 diffractometer
control equipped with Philips PW1728 X-ray generator. The scanning range was 3–20⁰ 2θ,
with a scanning rate of 1.2⁰ 2θ/min. Scanning electron microscopy images were obtained
with a Zeiss EVO series scanning electron microscope (EVO 50) with resolution of 2.0 nm at
30 kV.
4.2.3 Water absorbency measurements
Each sample weighing 0.1 g (particle size 100–240 mesh size) was immersed in the excess of
distilled water (pH 7.0, EC 0.001 mhos/cm) in triplicate and kept at two temperatures, 25⁰
and 50⁰C, until equilibrium was attained. Free water was filtered through a nylon sieve (200
mesh size), gel allowed to drain on sieve for 10 min, and finally weighed. The water
absorbency (QH₂O) was calculated using the equation: QH₂O (g/g) = (w₂-w1)/w1, where w1 is the
weight of xerogel (dry absorbent) and w2 is the weight of swollen gel. QH₂O was calculated as
grams of water per gram of dry sample. The GG-SAP exhibiting maximum absorption in
distilled water was further evaluated in different salt solutions.
4.2.4 Salt solution absorbency measurements
Vacuum dried GG-SAP exhibiting maximum QH₂O in distilled water was milled to achieve
particle size in the range of 100–240 mesh size. Aqueous solutions of different strengths (5,
10, 15 and 20 mM) of four salts, ammonium sulphate (AS), ammonium nitrate (AN),
potassium nitrate (PN), sodium chloride (SC), and one fertilizer, urea (U) were prepared and
used. The dried and milled sample (0.1 g) was immersed in salt solution of a particular
strength at 50⁰C. The swollen gel was weighed after 24 h and QH₂O was calculated using same
equation as above. Another similar experiment was repeated in tap water (pH 7.7, EC 2.04
mhos/cm), hard water, and aqueous solutions of pH 4, 7, and 9. Hard water of three different
strengths was prepared in the laboratory according to CIPAC standard method and labelled as
hard water A (hardness 20 ppm, pH 5–6, and Ca: Mg ratio 50: 50), hard water B (hardness 20
30
ppm, pH 8–9, and Ca : Mg ratio 80 : 20), and hard water C (hardness 500 ppm, pH 7–8, and
Ca : Mg ratio 80 : 20).
4.2.5 Water absorption and retention measurement in soil and soil-less media
Sandy loam soil (pH 7.8 measured at 1:1.25 soil to water ratio; organic carbon (OC) content:
0.51% for natural soil (determined by the Walkley and Black method) (Jackson, 1967); soil
mechanical fractions: sand 78%, silt 10%, clay 12.4%, employing the Bouycos hygrometer
(Black et al., 1965) method: cation exchange capacity 11.2 Cmol kg-1
by the normal
ammonium acetate (Jackson, 1967) (pH 7.0) method) and soil-less media (a sterilized mixture
of coco peat, vermiculite, and perlite in the ratio 3: 1: 1 on volume basis) were collected from
institute farm and protected cultivation nursery, respectively. Air-dried soil sample was
passed though the 2-mm sieve and mixed with GG-SAP at the rate of 0.5% and 0.75% each.
Soil-less medium was dried till constant weight was attained and used as such. For water
absorbency measurement, desired amounts of amended soil (50 g) or soil-less medium (20 g)
were taken separately in pre-weighed plastic cups having perforated bases fitted with filter
papers. Subsequently, each cup was immersed overnight in water to saturate. Water held by
the sample was determined gravimetrically. Water holding capacity (WHC) of soil and soil-
less medium was calculated by the equation: WHC (%) = M-m/ m×100, where M denotes
weight of wet sample (g) and m denotes weight of oven-dried sample (g).
Water retention study was done using pressure plate apparatus at different tensions
(2.3 pF, 2.8 pF, 3.0 pF, 3.7 pF, 4.0 pF and 4.2 pF). Ceramic plates were kept overnight in
water for saturation. Amended soil and soil-less media were filled in rubber rings arranged on
bar plates and allowed to saturate overnight. The saturated samples along with ceramic plates
were placed in pressure chamber pertaining to different tensions. Pressure was applied and
maintained till water stopped flowing out of the chamber. Dehydrated moist and finally
samples were transferred to moisture boxes immediately and weighed. The moist samples
were dried in a hot air oven at 105⁰C for 24 h, air cooled, and reweighed. The amount of
water held at particular pressure was calculated using following equation:
WC (% w/w) = [(Wwet – Wdry)/ Wdry] × 100
Where WC is the percent water content of soil or soil-less media on weight basis,
Wwet is the mass of wet soil or soil-less media at a particular tension, and Wdry is the weight of
oven-dried soil or soil-less medium.
31
4.2.6 Statistical analysis
The experiments on effect of various parameters on water absorbency values were conducted
using factorial completely randomized design. To identify the best treatment combinations,
the data were analysed by one-way classified analysis using PROC GLM procedure of SAS
package (SAS Institute, Cary, NC).
4.3 Results and discussion
4.3.1 Synthesis technique selection: comparison of microwave and thermally initiated
polymerization.
Table 1: Swelling response of GG-SAP prepared by conventional and microwave techniques
Monomer wt.% Conventional Microwave
Q H₂O (g/g)
2.5 154.9
2.6
3.75 492.3
4.8
5.0 464.2
9.5
7.5 423.4
56.7
10 394.3
125.6
15 354.4
189.6
It is clear from Table 1 that under the chosen experimental conditions, microwave assisted
synthesis resulted in hydrogels with inferior swelling ratios as compared to the conventional
technique. This observation is contrary to one reported by Sen et al, in (2010). As compared
to the conventional technique, the microwave assisted synthesis exhibited continuous
increase in swelling ratio of generated hydrogels. Sen et al, (2010) reported threshold
monomer content of approximately 15 wt %, which is the highest monomer content in the
present work. Therefore, microwave assisted technique may not be an economical option for
the synthesis of guar gum based hydrogels.
32
4.3.2 Characterisation of superabsorbent hydrogels
4.3.2.1 FTIR analysis
The FTIR spectra of native guar gum, acrylamide and cross linked guar gum-g- polyacrylate
hydrogel are shown in Figure 1.
Figure 1: FT-IR spectra of guar gum, acrylamide and GG-SAP
The absorption bands of Guar gum (GG) at 1661 cm-1
assigned to H-OH bending and at 1439
cm-1
assigned to C-OH bending vibration almost disappeared in FTIR of the hydrogel (Figure
1), where new bands at 1654 cm-1
(C=O stretching of COO- groups), 1570 cm
-1 (asymmetric
stretching of COO-) and at 1411 cm
-1 (symmetric stretching of COO
-) appeared. Bands of GG
at 1020 cm-1
(C-O stretching), 1082 (C-OH stretching) and 1158 cm-1
(C-O-C stretching),
although are visible in the spectrum of hydrogel, the intensity is weaker. Shift of ≈30 cm-1
towards longer wavelength in the O-H stretching band in hydrogel can be attributed to inter/
intramolecular H-bonding. Absence of all the characteristic peaks of CONH2 group of
acrylamide (3361 cm-1
(υa NH2), 3198 (υs NH2), 1670 (υ C=O), 1429 (υ C-N), 1352 ($ NH2),
1280 (γ NH2), 961 (ω NH2) and appearance of the characteristic peaks of COOH and COO-
group as described above, confirm successful conversion of amide into COO-
moiety.
Tentative scheme of formation of developed SAPs is depicted in Figure 2.
33
Figure 2: Diagrammatic representation of formation of a typical cl-gg-g-(polyacrylate)
hydrogel
4.3.2.2 Solid state C13
NMR analysis
Figure 3 depicts C13
NMR spectrum of an optimised cross linked GG-g- polyacrylate
hydrogel. NMR peaks in the spectrum were compared with the peaks of native guar gum
(Cheng et al., 2012). Signal at 185.7 ppm can be ascribed to the COO- group Cs in the grafted
34
polyacrylate network. This peak is not visible in the spectrum of guar gum. Assignments
characteristic of the guar gum (Kriz et al., 2001) are clearly visible in the spectrum of the
hydrogel and are as follows: 108.8 ppm (C-1 of galactose and mannose units), 99.88 ppm (C1
of β- D- mannose reducing chain end), 73.39 ppm (C2- C5 of galactose and mannose) and 64
ppm broad peak (C6 of galactose and mannose units in Table 2). Characteristic peak of
methylenic C linking two amide groups in the cross linker bisacrylamide at 45.9 ppm is
visible in the spectrum of GG-SAP. Absence of peaks pertaining to olefinic Cs in spectra of
parent monomer and cross linker confirms free radical initiated grafting and cross linking of
polyacrylate network. Additional peaks at 40.30 to 45.97 ppm can be assigned to various Cs
(CH2-CH2-) of the polacrylate network grafted on to the guar gum. Absence of the peaks in
this region in the back bone’s spectrum confirms in situ grafting and cross linking.
Table 2: Assignment of various peaks in C13
NMR spectrum of guar gum (Cheng et al.,2012)
Structure C-1 C-2 C-3 C-4 C-5 C-6
α-D-Gal 101.7 71.3 72.1 72.3 74.02 63.9
β-D-Man
unbranched
at O-6 (U)
102.9 72.8
72.84
74.25
74.34
79.26
79.51
77.98
77.92
77.85
63.48
β-D-Man
branched at
O-6 (B)
102.80 72.84
72.7
74.2
79.51
79.73
76.34
72.73
69.8
69.68
69.51
β-D-Man
non-
reducing
chain end
102.35 72.89 75.80 69.51 78.40 63.48
β-D-Man
reducing
chain end
96.55 73.29 74.34 79.26 77.9 63.48
4.3.2.3 Morphological SEM
Surface morphology of cross linked GG-g- polyacrylate hydrogel vis-à-vis native guar gum
was examined under scanning microscope to investigate the effect of grafting and cross
linking on the surface morphology of the back bone polymer.
35
Figure 3: C13
NMR spectra of A) Monomer B) Cross linker C) GG-SAP
Figure 4 compares the morphology of GG (SEM images reproduced from Gong et al., 2011)
vs. GG-SAP. SEM photograph of guar gum displays dense and smooth surface of the back
bone Figure 4, as is evident from grafting and cross linking of polyacrylamide chains on to
the back bone resulted in homogeneous but fractured topography. Another noteworthy feature
in the SEM micrographs is uniform porous appearance on the surface of the hydrogel. In
most of the previous work done on guar gum-g- Polyacrylate hydrogels, non-porous, uniform
morphology has been reported which is caused by incorporation of clay minerals (Wang et
al., 2010).
36
Figure 4: SEM images of guar gum (A and B) (Gong et al., 2011) and representative GG-
SAPs (C-D)
In contrast to this, hydrogel prepared in the present study exhibit ragged and porous
topography. This observation highlights the impact of the reaction conditions and drying
techniques used in the preparation of hydrogels. Porous morphology of hydrogels in our work
can be attributed to the solvent assisted drying technique. This fact has been confirmed in our
previous studies also (Anupama et al., 2005).
4.3.2.4 XRD analysis
X-ray diffraction patterns of native guar gum and GG-SAP are presented in Figure 5. The
XRD peaks of the both the materials confirms their amorphous character. A very low degree
of crystallinity in guar gum is indicated by its characteristic diffraction band (2θ = 20⁰). XRD
of GG-SAP also showed major diffraction band at the same position (2θ = 20⁰) indicating
A B
C D
37
that grafting and cross linking of polyacrylate chains onto the guar gum back bone did not
influence the crystallinity of the later. However as is evident from Figure 4, an addition peak
(2θ = 9⁰), appears in the XRD of GG-SAP which can be ascribed to the lateral bilayering of
the polyacrylate chains.
Figure 5: XRD peaks of acrylamide, guar gum and GG-SAP
4.3.3 Effect of monomer to back bone ratio on water absorbency
The effect of different monomer to backbone ratios recorded on water absorbency of
prepared hydrogels is shown in Table 2. It can be seen that initially the equilibrium water
absorbency increased with increase in monomer content up to monomer backbone ratio of
0.75:1. Further increase up to investigated ratio of 3:1, the QH₂O exhibited consistent fall.
Initial increase in amount of acrylamide leads to increase in fraction of polyacrylamide
chains, containing sufficient number of polar –CONH2 groups. The electrostatic repulsion up
to this point seems to favour the water absorption characteristics of the polymer. Decrease of
QH₂O beyond weight ratio 0.75:1 can be attributed to increase in homopolymer content,
resulting in increase in soluble fraction at fixed cross linker and initiator amount in the feed
(Singh et al., 2011; Zhang et al., 2006). In a related work on GG-g- polyacrylates reported by
Wang et al., (2010) similar trend was reported; though the maximum equilibrium water
38
absorbency was achieved at much higher monomer content in the feed. The variations can be
ascribed to the difference in reaction process conditions reported earlier and in the present
study.
4.3.4 Effect of cross-linker and initiator concentration on water absorbency
The effect of cross-linker content on water absorbency behaviour of prepared hydrogels is
shown in Table 3. It is clear from the table that at a fixed monomer and initiator
concentration, the swelling ratio exhibited significant increase with cross linker concentration
increase in the range 1.0 wt. %, beyond which it significantly decreased with increase in cross
linker concentration. The initial increase may be understood in terms of decrease in
homopolymer and soluble fractions in the feed due to expansion in cross linked network. Fall
in water uptake above 1.0 wt. % can be easily explained in terms of Flory’s theory (Flory,
1953), according to which a high concentration will induce extensive crosslink points,
resulting in increase of cross linking density. As a result, the network relaxation and the voids
for holding water decrease leading to decrease in water absorption. Under experimental
conditions chosen in the present study, concentration of the cross linker should be higher than
0.03 wt. %, otherwise gel setting cannot be achieved. This can be understood in terms of the
insufficient number of cross linker molecules need to graft and create stable polymer
network. Guar gum and polyacrylate though are hydrophilic in nature, lack of proper cross
linking leads to sol-gel states in water and QH₂O cannot be measured.
The amount of initiator, as can be seen from Table 3, plays significant role in the graft
polymerization reaction. It is evident that under constant reaction conditions, at concentration
lower than 3.0 wt. %, the water absorbency values are lower. Low initiator concentration
slows down the rate of graft polymerization and generation of polymer network on to the
back bone. After attaining optimum structural characteristics favourable for maximum water
absorption, further increase in the concentration of initiator in the present study >3.0 wt. %,
the reaction velocity increases, resulting in smaller networks of low molecular weights.
Relative ratio of free polymer chain ends that do not contribute to water absorption increases
(Chen and Zhao, 1999), and thus the decrease of water absorbency of the hydrogels are
observed at higher concentrations of initiator.
4.3.5 Effect of volume of water in the feed on water absorbency
39
The variation of QH₂O with quantity of water in the feed mixture, expressed as ml/g reaction
mass, is shown in Table 4. It is evident that increase in quantity of water from 2.8 to 11 g/ ml
of feed mass showed consistent increase in the QH₂O values of hydrogels. An optimum
network formation requires effective collisions between various reaction moieties, for which
water plays significant role as reaction medium. At volume less than 11 ml/g, low QH₂O
values are due to tightly cross linked hydrogel network formation. Increase of VH₂O beyond
11 ml/g leads to increase in soluble fraction due to dilution of cross linker, thus leading to
poor network formation. This fact is reflected in the decrease in water absorbency on increase
of VH₂O from 11 ml/ g to 16.5 ml/ g under our experimental conditions. These findings have
been also described in our previous study (Singh et al., 2011).
Table 3: Effect of monomer to back bone ratio, cross-linker and initiator concentration on the
water absorption of GG-SAP
Monomer
concentration1
(wt.%) Q H₂O (g/g)
Cross linker
concentration2
(wt.%)
Q H₂O (g/g) Initiator
3
(wt.%) Q H₂O (g/g)
2.5 154.9f 0.05 48.2
f 0.03 47.8
g
3.75 492.3a 0.1 192.8
e 0.06 182.4
f
5 464.2b 0.5 490.5
b 0.3 320.3
e
7.5 423.4c 1.0 513.1
a 0.6 354.6
d
10 394.3d 5.0 449.2
c 3.0 507.8
a
15 354.4e 10.0 350.8
d 4.5 482.4
b
5.0 421.9
c
5.5 404.2d
LSD at 5% 6.96 10.07 7.07
CV 1.0064 1.6259 1.2710
F value 2990.48 3351.77 5504.92
Means within a column followed by different letters are significantly different at 5% level of significance and
those following by the same letter do not significantly at 5% level of significance. 1Synthesis parameters: weight ratio of cross linker and initiator to backbone: 0.005:1 and 0.05:1, respectively, V
H₂O 11.42 ml/ g feed. 2Synthesis parameters: weight ratio of monomer and initiator to backbone: 0.75:1 and 0.05:1, respectively, V H₂O
11.42 ml/ g feed. 3Synthesis parameters: weight ratio of monomer and cross linker to back bone: 0.75: 0.01:1 and 0.05:1,
respectively, V H₂O 11.42 ml/ g feed.
40
4.3.6 Effect of molar ratio of alkali to monomer on water absorbency
According to Flory’s network theory, the fixed charges on the polymer network contribute
significantly to the swelling of superabsorbent. The effect is excreted by these charges
through electrostatic repulsion and osmotic potential difference between the network and
surrounding solution. Therefore the type and amount of hydrophilic groups are important in
determining fluid absorption are important in determining fluid absorption properties of
hydrogels. The effect of molar ratio of alkali to acrylamide on equilibrium water absorbency
of GG-SAP in distilled water is shown in Table 4, the QH₂O increases with increase in molar
ratio from 0.5 to 2.0 under experimental conditions of the present study. Further increase led
to fall in the QH₂O. On saponification of polyacrylamide by NaOH, the ionic hydrophilic
moieties in polymer structure increase that cause increase in osmotic potential difference and
the electrostatic repulsion causes disentanglement of polymer chains resulting in expansion of
the network.
Table 4: Effect of water volume and molar ratio of alkali and monomer on water absorbency
of GG-SAP
Water volume4
(ml/g) Q H₂O (g/g) Molar ratio of NaOH
to Acrylamide5
Q H₂O (g/g)
2.84 88.2f
0.5 178.3f
5.68 195.1e
1.0 354.9e
8.52 351.7c
1.5 426.9d
11.36 517.1a
2.0 518.4a
14.2 434.2b
2.5 498.3b
16.5 288.4d
3.0 467.2c
LSD at 5% 7.39 6.91
CV 1.2376 0.9431
F value 5943.34 3889.66
Means within a column followed by different letters are significantly different at 5% level of significance and
those following by the same letter do not significantly at 5% level of significance. 4Synthesis parameters: weight ratio of monomer, cross linker and initiator to backbone: 0.75: 0.01:1 and 0.05:1,
respectively. 5Synthesis parameters: weight ratio of monomer, cross linker and initiator to backbone: 0.75: 0.01:1 and 0.05:1,
respectively, V H₂O 11.42 ml/ g feed.
4.3.7 Effect of particle size of backbone polymer on the water absorbency
As seen in Table 5, decrease in particle size of guar gum used in hydrogel preparation up to
100- 240 mesh led to increase in the swelling ratio under experimental temperature (50⁰ C).
41
Finer particles of size >240 mesh size led to formation of hydrogels with reduced QH₂O. As
described previously (Singh et al., 2011), an increase in specific surface area of back bone
with decrease in particle size attribute in particle size attribute to the superior networks with
optimum crosslink density. Hydrogels prepared from guar gum of particle size >240 mesh
size exhibit reduced water absorbency probably due to inability of initiation to generate
sufficient free radicals on the highly expanded backbone chains density.
Table 5: Effect of particle size of back bone on water absorbency of GG-SAP
Particle size (mesh) Q H₂O (g/g)
25-100 484.4c
100-240 603.2a
>240 516.9b
LSD at 5% 17.18
CV 1.4173
F value 196.61
Means within a column followed by different letters are significantly different at 5% level of significance and
those following by the same letter do not significantly at 5% level of significance.
Synthesis parameters: weight ratio of monomer, cross linker and initiator to backbone: 0.75: 0.01:1 and 0.05:1,
respectively, V H₂O 11.42 ml/ g feed.
4.3.8 Effect of temperature and pH on swelling
In our previous studies (Singh et al., 2011) and studies done by many workers (Li et al.,
2005; Suo et al., 2007), the equilibrium swelling of hydrogels in water was found dependent
on the temperature. As is clear from Table 6, the QH₂O increases significantly with increase in
temperature from 10⁰ to 55⁰ C. Temperature in the range of 25-50⁰ C are of particular interest
to assess the suitability of hydrogels in agriculture.
As in seen in Table 6, QH₂O of anionic GG-SAP exhibited significant response as a
function of pH of swelling medium. GG-g-polyacrylate hydrogels contain hydrophilic -
COOH and -COO-
groups which ionize further at pH 9.0 and exert electrostatic repulsion
leading to expansion of polymer network and hence the increase in QH₂O. Similar finding has
been described by (Kiatkamjornwong and Phuncharcon, 1999). Comparatively lower water
absorbency at pH 4.0 can be attributed to non-ionization of –CONH2 groups and –COOH
groups. Our laboratory has developed a cellulosic superabsorbent, P-gel (Anupama et al.,
2005), which has been established as water retentive aid in agriculture. However its use in
problem soils (acidic and alkaline) is restricted due to its low QH₂O values in acidic and
42
alkaline pH environment. Superior swelling characteristics of GG-SAP at pH 4- 9 and more
particularly 9, point towards a potential soil conditioner for the acidic and alkaline soils.
4.3.9 Effect of quality of water on absorbency
Water quality plays an important role in determining the performance of
superabsorbents in agriculture under most practical use conditions; hard water is available for
agricultural use. The effect of water quality on the swelling ratio of GG-SAP is shown in
Table 6. As compared to maximum QH₂O values of the product obtained in distilled and
deionized water, the swelling in tap water (EC = 2.04 mhos/ cm, pH = 7.7), hard water of
different simulated ionic strengths though decreased, but not drastically in comparison with
our earlier reports on swelling behaviours of P-gel and biopolymeric superabsorbent
nanocomposite (Singh et al., 2011). Decrease of water absorbency with increase in its ionic
strength of hard water may be explained in terms of screening of –COO- anions by the cations
present in water. Similar observation has been described by Pourjavadi et al. (2008).
Table 6: Effect of temperature, water quality and pH on swelling of GG-SAP
Temperature
(⁰C)
Q H₂O (g/g) Water quality Q H₂O (g/g) pH QH₂O (g/g)
10 490.7e
Deionized water 675.8a
25 519.2d
Distilled water 611.9b
4.0 466.8c
35 542.4c
Tap water 452.2c
7.0 669.2b
45 593.8b
Hard water- A 361.0d
9.0 744.3a
50 621.1a Hard water- B 250.3
e
Hard water- C 215.6f
LSD at 5% 3.39 6.54 5.82
CV 0.3205 0.8407 0.4102
F value 4915.60 8211.97 9349.45
Means within a column followed by different letters are significantly different at 5% level of significance and
those following by the same letter do not significantly at 5% level of significance.
4.3.10 Effect of time period on water absorbency
As can be seen from Figure 6, water absorption rates of the optimised GG-SAP, were
measured at 25⁰ C and 50⁰ C. Results shown in Figure 5, indicate that the GG-SAP attains
43
equilibrium water absorbency in 14 hr at 50⁰ C, whereas at 25⁰ C the same is attained in 18
hours. Swelling of superabsorbent polymer involves large segmental motion that further
results in increased expansion of macromolecular chains (Bajpai & Johnson, 2005).
Buchanan et al. (1986) suggested that swelling rate of hydrogel is a function of
chemical structure, surface area, particle size and density of the polymer. Initially low
swelling rates at both the temperatures may be explained in terms slow diffusion and
restricted capillarity in the initial polymer network. The absorbency rates of biopolymeric
hydrogels can be significantly improved by addition of hydrophilic clays and through
generation of porosity (Wang et al., 2009).
Figure 6: Effect of time period on swelling at 50⁰ C and 25⁰ C
4.3.11 Effect of salt type and strength on water absorbency
Evaluation of the swelling behaviour of GG-SAP in salt solutions, particularly those applied
as fertilizers and present in saline soil, is very important in view of their agricultural and
horticultural applications. Figure 7 depicts the comparative swelling response of the
optimized GG-SAP in solutions of varying strengths of (NH4)2SO4, NH4NO3, KNO3, NaCl,
and urea. In all the salt solutions, absorption was less compared with that in distilled water.
The hydrogel exhibited minimum reduction in QH₂O in urea solutions at all test
concentrations. This observation is of interest because for most of the crops in agriculture,
urea comprises an important agro-input. In general, decrease in QH₂O with increase in
44
concentration of salt solutions may be understood in terms of reduction in osmotic pressure
difference between gel matrix and external salt solution.
4.3.12 Effect of hydrogel addition on water absorption and retention capacity of plant
growth media (sandy loam soil and soil-less medium)
Water absorption capacity of the test sandy loam soil and soilless medium significant
increased on addition of GG-SAP hydrogel. As is clear from the Table 7, at both the
experimental temperatures (25⁰ C and 45⁰ C), the hydrogel amended soil and soilless medium
absorbed more water than respective controls. Amendment with hydrogel @ 0.75% exhibited
significantly higher water absorbency (WAC) than @ 0.5% in both the plant growth media.
Figure 7: Effect of salt/ fertilizer type and their strength on water absorbency
In case of soil, the amended samples at 50⁰ C exhibited higher WAC than at 25⁰ C at both
0.5% and 0.75% amendment levels. Similar pattern, though less pronounced was exhibited by
the amended soilless medium. GG-SAP exhibits increase in QH₂O with increase in temperature
due to expansion of the polymer network as described earlier and the same behaviour is
manifested in the amended media also. Similar observation was described in our earlier
reports on biopolymeric hydrogels (Anupama et al., 2005).
Utility of the developed superabsorbent material (GG-SAP) for agricultural and
horticultural applications was evaluated in terms of its effect on the moisture characteristics
of the plant growth media under different matric tensions. As compared to control, the water
holding capacity (WHC) of the hydrogel (GG-SAP) amended soil, at both rates 0.5% and
45
0.75% remained higher at field capacity and also at all matric tension respectively, though the
difference between 0.5% and 0.75% amended soils became narrower at matric tensions above
2.53 pF. Similar behaviour was observed for the soilless medium. The hydrogel (GG-SAP)
amended soilless medium @ 0.5% and 0.75% showed higher WHC values (65.2% and
85.29%) respectively, as compared to control. Irrespective of the type of absorbent material,
the percent moisture absorbed by free SAP is more relative to when it is present in a plant
growth medium. This is because in such situations, the gel particles are surrounded by the
soil or media particles, which subject it to confirming pressure, (Yangyuoru et al., 2006) and
limit the degree of swelling.
Table 7: Water absorbency behaviours of GG-SAP in soil and soilless medium at 25⁰ C and
50⁰ C
Hydrogel
content
(wt.%)
Soil
Hydrogel
content
(wt.%) Soil less medium
25⁰C 50⁰C 25⁰C 50⁰C
0.0 42j
41.5j
0.0 283.3e
284.6c
0.5 94.9i
112.7h
0.5 402.3d
428.1c
0.75 145.4g
186.7f
0.75 454.9b
476.1a
LSD at 5% 6.39
CV 1.4922
F value 5884.59
Means within a column followed by different letters are significantly different at 5% level of significance and
those following by the same letter do not significantly at 5% level of significance.
Water harvested by the hydrogel should be readily available to plant, thus avoiding water
stress and enable its survival (Choudhary et al., 1995). Thus is expressed in terms of the
available water capacity (AWC) or readily available water (Figure 8) that refers to difference
between field capacity and soil moisture at a threshold value of matric tension that calculated
the following expression (Singh, 2004):
Available Water Capacity (AWC) = (FC- PWP).ρ/ 100,
Where FC is field capacity; PWP is permanent wilting point; ρ is bulk density of media used
Addition of GG-SAP to soil and soilless medium increased water availability to plant
as compared to respective control. In case of soilless medium, the values of readily available
46
water (RAW) at both amendment levels whereas in case of soil amended @ 0.75% was able
to make more water available. As is clear from Figure 9, compared to control, water holding
capacity (WHC) of soil and soilless medium amended with the GG-SAP remained higher
with rise in matric tension. At field capacity, in both soil and soilless media, hydrogel
addition led to higher percent retention as compared to the unamended controls.
Figure 8: Effect of gel addition on available on water from soil (A) and soil less medium (B)
Moisture release curves (Figure 10, A and B, expressed as moisture cm cm-1
of the hydrogel
amended soil and soilless media show that as compared to individual controls, the gel
amended soil and soilless media exhibited significantly higher release of moisture. Moisture
release was calculated follows (Ritchi et al., 1972):
(MP-Mo).ρ / 100
Where Mp is moisture held at particular pressure, Mo is moisture held at zero
pressure. ρ is the bulk density of soil or soilless medium. At 4.2 pF (corresponding to
47
permanent wilting point) hydrogel amended soil released (21 to 37%) more water than
control. Similar behaviour was observed in case of soilless medium. Relative moisture release
was higher in soilless medium as compared to the soil at both amendment levels. This can be
attributed to the loose texture of the soilless medium.
Figure 9: Moisture retention curves of soil medium (A) and soilless media under different
matric tensions (pF) in unamended and gel amended condition
4.4 Conclusion
Probability of scale up of a laboratory process depends up on its simplicity and ease of
handling. Emphasis in our lab has always been to develop agricultural and eco-friendly and
economic soil conditioners as water retentive aids in agriculture. The hydrogels developed in
the present study has exhibited all the characteristics that qualify it as a potential candidate
for agricultural use. As compared to earlier soil conditioners developed by us, GG-SAP
possess additional advantage and merits in terms of superior water absorbency behaviour
under acidic and alkaline conditions, in the presence of salts and fertilizers and the moisture
retention and release characteristics imparted to the plant growth media. Guar gum, being a
48
biopolymer with hydrophilic biocompatible characteristics, the hydrogels based on it will be
exploited as potential bioformulations. The study showed that the backbone monomer ratio,
cross linker concentration and quantity of water per unit reaction mass were critical
parameters in determining the swelling properties. Structural characterization confirmed the
successful formation of the hydrogels based on guar gum.
Figure 10: Moisture release curves of soil medium (A) and soil less medium (B) under
different matric tensions (pF) in unamended and gel amended condition
49
RESEARCH PAPER -II
Novel cross linked guar gum-g-poly(Aam) porous superabsorbent hydrogels:
characterization and swelling behaviour in different environments
Abstract: A series of cross linked guar gum-g-poly(Aam) was prepared by in situ grafting
polymerization and cross-linking on to a natural guar gum. The optimization of various
synthesis parameters w.r.t the water absorption capacity of the hydrogels (GG-SPH) was
done. GG-SPHs were characterized by X-ray diffraction, scanning electron microscopy, FT-
IR and solid state C13
NMR spectroscopy. Swelling behaviour of a candidate hydrogel (GG-
SPH) in response to external stimuli namely, salt solutions, fertilizer solutions, temperature,
and pH was studied. The hydrogel exhibited significant swelling in various environments.
Effect of cross linker concentration on the porosity and density of porous hydrogels exhibited
decrease in porosity and increase in density. Swelling of the GG-SPH was faster than the
corresponding GG-SAP. SEM analysis confirmed porous morphology of the prepared
hydrogels.
Key words: Guar gum; porous; swelling
5.1 Introduction
Hydrogels are cross-linked hydrophilic polymers with a network structure consisting of
acidic, basic, or neutral monomers which are able to imbibe large amounts of water (Omidian
et al., 2002). Because of their network structures and the possibility of rearrangements of
hydrophobic/hydrophilic domains during the swelling process, including entanglements and
crystalline regions, these polymers are water insoluble (Choi et al., 2007). Basically
hydrogels are of two types with respect to swelling: conventional hydrogels and new
generation of hydrogels (Chavda et al., 2010). The most important difference between these
two groups is their swelling characteristic carbons. The swelling properties of hydrogels are
mainly related to the elasticity of the network, the extent of cross-linking, and porosity of the
polymer (Kim et al., 2004). Among the new generation hydrogels, superporous hydrogels are
lightly crosslinked hydrophilic polymers that can absorb, aqueous solutions up to hundreds of
times their own weight in shorter duration (Hyojin et al., 2006).The fast swelling of these
polymers can be related to capillary wetting of interconnected open pores (Yin et al., 2007).
50
Of late, biopolymeric superporous hydrogels are of interest due to their multifarious
applications in domains such as drug delivery and tissue engineering etc. (Chen et al., 1999).
Because of their biocompatibility, biodegradability and non-toxicity, polysaccharides and
protein-based porous hydrogels have created extensive interest (Omidian et al., 2006). Guar
gum, a non-ionic galactomannan polysaccharide seed gum derived from Cymaposis
tetragonolobus, has also been used in discrete studies to develop SAPs and SAP composites
with pH-sensitive characteristics(Zhang et al., 2006). Their use as backbone to prepare
porous hydrogels in still not reported to the best of our information. In the present article,
guar gum-g-poly(Aam) porous superabsorbent hydrogels have been reported. Reaction
variants namely cross linker concentration; initiator concentration, volume of water per unit
feed etc. influence the swelling characteristic carbons and network properties of hydrogels. In
most of available reports, such extensive investigations are lacking for porous hydrogels. The
effects of various reaction parameters namely cross linker concentrations, initiator
concentration, volume of water per unit feed, nature of foam stabilizers and foaming aids and
their concentrations, duration of reaction etc. has been presented in detail and supported by
structure characterisation. In the second part of the study, swelling behaviour of the prepared
SPH as a function of external stimuli namely, pH, salts and fertilizers and water quality has
been discussed.
5.2 Materials and methods
5.2.1 Chemicals
Commercial guar gum (purity 95%), acrylamide, N,N’- methylenebisacrylamide, N,N,N’,N’
tetramethylenediamine (TEMED) and sodium bicarbonate were purchased from SD Fine
(Pvt) Ltd., Mumbai, India. Persulphate initiator, surfactants (FS-1, FS-2, FS-3, FS-4) and
foaming aids (OA-1, OA-2, OA-3 and OA-4) were purchased from Merck, (Pvt) Ltd.
Mumbai, India. All the reagents were used as such without further purification.
5.2.2 Synthesis of superporous cl-GG-g-poly(Aam) hydrogels
The SPH hydrogels were prepared by free radical polymerization technique employing
persulphate-TEMED redox initiator system. A general method of preparation followed in
earlier reports (Chen et al., 1999; Guo and Gao, 2007; Park et al., 1993), involves sequential
addition of monomer, cross linker, initiator, foam stabilizer and the foaming aid in distilled
water under inert atmosphere at 70-80⁰ C (Figure 1). In the present study, six different
51
sequences of addition of reactants were tried, details of which will be protected under IPR. A
typical procedure comprised dissolution of all the reagents including the backbone in distilled
water. pH was adjusted and the reaction was initiated by the persulphate-TEMED redox pair.
The time for harmonizing gelation and foaming reactions was standardised. The solution was
kept undisturbed for a specific period. The resultant SPH were washed, dehydrated and
finally dried in hot air oven.
Figure 1: General steps involved in SPH synthesis
The hydrogels were characterized by FT-IR spectra recorded in KBr disc carbons on a Bruker
Fourier Transform Infrared Spectrophotometer under dry air at room temperature. Solid state
C13
NMR spectra of optimized GG-SPH were recorded on a BRUKER DSX 300 instrument
operating at 7.04 tesla, carbon frequency 75.47 MHz for carbon. Cross linker and monomer
were characterized by liquid state C13
NMR using BRUKER Avance instrument. Scanning
electron microscopy images were obtained with a Zeiss EVO series scanning electron
microscope (EVO 50) with resolution of 2.0 nm at 30 kV.
5.2.3 Water absorbency measurements
A sample weighing 0.1 g (particle size 100–240 mesh size) was immersed in the excess of
distilled water (pH 7.0, EC 0.001 mhos/cm) in triplicate and kept at two temperatures, 50⁰C,
until equilibrium was attained. Free water was filtered through a nylon sieve (200 mesh size),
gel allowed to drain on sieve for 10 min, and finally weighed. The water absorbency (QH₂O)
was calculated using the following equation: QH₂O (g/g) = w₂-w1/w1; where w1 is the weight of
xerogel (dry absorbent) and w2 is the weight of swollen gel. QH₂O was calculated as grams of
water per gram of dry sample. The SPH exhibiting maximum absorption in distilled water
was further evaluated in different salt solutions.
52
5.2.4 Salt solution absorbency measurements
SPH was milled to achieve particle size of the range 100–240 mesh size. Aqueous solutions
of different strengths (5, 10, 15, and 20 mM) of four salts namely ammonium sulphate (AS),
ammonium nitrate (AN), potassium nitrate (PN), sodium chloride (SC), and fertilizer namely
urea (U) were prepared and used. The dried and milled sample was immersed in salt solution
of a particular strength at 50⁰C. The swollen gel was weighed after 24 h, using same equation
as above. Similar experiment was repeated in tap water (pH 7.87, EC 1.099 mhos/cm), hard
water, and aqueous solutions of pH 4, 7, and 9. Hard waters of three different strengths were
prepared in the laboratory according to CIPAC standard method (Daasch, 1947) and labelled
as hard water A (hardness 20 ppm, pH 5–6, and Ca : Mg ratio 50 : 50), hard water B
(hardness 20 ppm, pH 8–9, and Ca : Mg ratio 80 : 20), and hard water C (hardness 500 ppm,
pH 7–8, and Ca : Mg ratio 80 : 20).
5.2.5 Density and Porosity measurements
For density measurement, the solvent replacement method was used (Tang et al., 2005). A
piece of dried SPHC was taken and weighed. It was immersed in a predetermined volume of
hexane in a graduated cylinder and the increase in hexane in a graduated cylinder and the
increase in hexane volume was measured as the volume of the polymer. The density was
calculated from the following equation: Density = MSPH/VSPH, where, VSPH is the volume of
solvent displaced by SPH and MSPH is the mass of the SPH. Experiment was done in
triplicate. For porosity measurement, dried SPHs were immersed in hexane overnight and
weighed after the excess hexane on the surface was blotted. The porosity was calculated from
the equation (Chavda et al., 2009): Porosity = VP/VT, where, VP = (VT - VSPH) is the pore
volume of SPH and VT is the total volume of the SPH. Total volume of SPH was measured
from its dimensions, as it was cylindrical in shape.
5.2.6 Statistical analysis
The experiments on effect of various parameters on water absorbency values were conducted
using completely randomized design. To identify the best treatment combinations, the data
were analysed by one-way classified analysis using PROC GLM procedure of SAS package
(SAS Institute, Cary, NC).
53
5.3 Results and Discussion
5.3.1 Characterisation of superporous hydrogel
5.3.1.1 FT-IR analysis
The FT-IR spectra of native guar gum, acrylamide and cl-gg-g-poly(Aam) (GG-SPH) are
shown in Figure 2. The absorption bands of Guar gum (GG) at 1661 cm-1
assigned to H-OH
bending and at 1439 cm-1
assigned to C-OH bending vibration almost disappeared in FTIR of
the hydrogel (Figure 2), where new bands at 1665 cm-1
(C=O stretching of COOH groups),
1435 cm-1
(O-H bending) and ≈ 1300 cm-1
(C-O stretching of carboxylic acid group appeared.
Bands of GG at 1020 cm-1
(C-O stretching), 1082 (C-OH stretching) and 1158 cm-1
(C-O-C
stretching), although are visible in the spectrum of hydrogel, the intensity is weaker. Shift of
≈30 cm-1
towards longer wavelength in the O-H stretching band in hydrogel can be attributed
to inter/ intramolecular H- bonding. Absence of all the characteristic peaks of CONH2 group
of acrylamide and appearance of characteristic of peaks of -COOH group confirm hydrolysis
of amide to –COOH group. During synthesis of SPH, it was observed that after addition of
NaHCO3 and onset of polymerisation pH of the medium increased to pH 12. This may
explain the conversion of -CONH2 to –COOH and –COO- groups.
Figure 2: FT-IR spectra of guar gum, acrylamide and GG-SPH
54
5.3.1.2 Solid state C13
NMR analysis
Figure 3C, depicts C13
NMR spectrum of an optimised cross linked porous hydrogel. NMR
peaks in the spectrum were compared with the peaks C13
NMR native guar gum given in
Table 1 (Cheng et al., 2012) and spectra of monomer (3A) and crosslinker (3B).
Figure 3: C13
NMR spectrum of monomer (A), cross linker (B) and GG-SPH (C)
Signal at 181.5 ppm can be ascribed to the COO- group carbons in the grafted polyacrylate
network. This peak is not visible in the spectrum of guar gum. Assignments characteristic of
the guar gum (Kriz et al., 2001) are clearly visible in the spectrum of the hydrogel and are as
55
follows: 103.6 ppm (C-1 of galactose and mannose units), 101.9 ppm (C1 0f β- D- mannose
reducing chain end), 72.6 ppm (C2- C5 of galactose and mannose) and 64 ppm broad peak (C6
of galactose and mannose units). Characteristic peak of methylenic C linking two amide
groups in the cross linker bisacrylamide at 43.3 ppm is visible in the spectrum of GG-SPH.
Absence of peaks pertaining to olefinic Carbons in spectra of parent monomer and cross
linker confirms free radical initiated grafting and cross linking of polyacrylate network.
Additional peak at 39.6 ppm can be assigned to (CH2-CH2-) Carbons of the polacrylate
network grafted on to the guar gum. Absence of the peaks in this region in the back bone’s
spectrum confirms in situ grafting and cross linking polymerization.
Table 1: Assignment of various peaks in C13
NMR spectrum of guar gum (Cheng et al.,
2012)
Structure C-1 C-2 C-3 C-4 C-5 C-6
α-D-Gal 101.7 71.3 72.1 72.3 74.02 63.9
β-D-Man
unbranched
at O-6 (U)
102.9 72.8
72.84
74.25
74.34
79.26
79.51
77.98
77.92
77.85
63.48
(broad)
β-D-Man
branched at
O-6 (B)
102.80 72.84
72.7
74.2
Split
79.51
79.73
76.34
72.73
69.8
69.68
69.51
β-D-Man
non-
reducing
chain end
102.35 72.89 75.80 69.51 78.40 63.48
β-D-Man
reducing
chain end
96.55 73.29 74.34 79.26 77.9 63.48
5.3.1.3 Scanning Electron Microscopy analysis
Morphology of a representative GG-SPH examined by SEM at various magnifications is
given in Figure 4. It is clearly visible from the images that the GG-SPH contains large
number of pores of different sizes. Dried hydrogel contains large number of pores of different
sizes. Pore size was estimated from the images by averaging the diameter of ten cells. The
average pore diameter calculated as 5.87 µm.
56
Figure 4: SEM images of nonporous GG-SAP (A) and porous GG-SPHs (B-C)
The images show that presence of guar gum and its participation in the polymerisation did
not destroy or affect the porous morphology of SPH. The SEM image of porogen free
hydrogel is shown in Figure 4, which appears nonporous in topography. In SEM of SPH, the
opaque surface surrounding the pores is assigned to the polysaccharide guar gum which is
very conspicuous due to high biopolymer-monomer ratios employed in the present study.
5.3.1.4 Elemental analysis
As compared to the backbone, higher content of C, H and N in GG-SPHs indicates grafting of
polyacrylamide/ polyacrylate chains on to the backbone. The data is present in Table 2.
Increase in the percent grafting with increase in monomer concentration is evident as the
percent C and N follows the order SPH-3> SPH-2> SPH-1.
5.3.2 SPH polymer synthesis and effect of reaction variables on water absorbency
behaviour
Major reactions steps involved in the preparation of SPHs are gelation and foaming. In order
to generate uniform pores in the matrix of the hydrogel, harmonization between foaming and
57
gelation is the key element. As described by Omidian et al., 2005, the whole process can be
divided into three stages (Figure 5). Stage- II involving synchronized gel and pore formation
is most critical. Therefore sequence of addition of various reactants plays determinant role in
the preparation of SPHs. A general synthetic procedure reported in most of the studies
(Omidian et al., 2005) is shown in (Figure 1).
Table 2: Elemental composition of superporous hydrogels
Figure 5: Typical harmonized gelation and foaming processes in the synthesis of
superporous hydrogels (Omidian et al., 2005)
For preparation of hydrogels evaluated in the present study, six methods differing in
the sequence of addition of various reagents and reaction conditions were studied. Details of
the methods will be covered under IPR. The effect of different methods on water absorbency
of the prepared SPHs is shown in Figure 6. As is clear from the Figure 6, SPHs from
methods-II, III and V exhibited statistically similar and superior absorbency in distilled water
as compared to I, IV and VI.
Material Nitrogen (%) Carbon (%) Hydrogen (%)
Back bone
SPH-1 (0.3% AM)
0.639
7.829
38.55
39.71
7.571
7.644
SPH-2 (0.5% AM) 11.67 41.79 7.649
SPH-3 (0.7% AM) 11.78 45.80 7.646
58
27.4
43.9
36.8
18.6
38.9
29.2
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
45.0
50.0
I II III IV V VI
QH
₂O (
g/g)
Method of preparation
Figure 6: Effect of method of preparation on water absorbency of GG-SPH, LSD at 5% degrees
of freedom is 5.22, CV is 8.8 and F value between treatments is 30.44. Synthesis parameters: weight ratio of
monomer, cross linker, initiator, foam stabilizer, porogen to backbone: 1.5:1, 0.01:1, 0.05:1, 0.1:1 and 0.1: 1,
respectively, V H2O 5.95 ml/ g feed.
R- COOH + NaHCO3
Foaming aid
R- COONa + CO2 + H2O
Figure 7: Scheme of reaction between foaming aid and sodium bicarbonate (SBC)
Cross linker plays an important role in influencing the SPH properties (Kabiri et al.,
2003). Effect of cross linker content on the water absorbency of SPHs is shown in Table 3.
As can be seen from Figure 7, foaming aid reacts with sodium bicarbonate, carbon dioxide
gas bubbles start evolving. Efficiency of the trapping the gas bubbles during gelation
determines the porosity of SPH. Increase in cross linker concentration favours high rate of
gelation. The increasing viscosity inhibits the CO2 bubbles to escape from the gel mass and
generates porous structure, which are manifested in the enhanced water absorbency. Similar
observation has been described by Kabiri et al (2003). In the present study, at cross linker
concentration >0.735 wt %, extensive rise in cross linking restricts the macromolecular
relaxation and the consequent rise in swelling.
59
Figure 8 shows that FS-4 behaves as the best foam stabilizer. Performance of a foam
stabilizer is determined by the length of duration up to which the foam is sustained (Chen and
Park, 2000). Effect of the concentration of the optimized foam stabilizer on water absorbency
is presented in Table 3. It is clear from the Table that increase in the concentration of foam
stabilizer from 0 wt % to 7.35% led to significant increment in the QH₂O values of the
corresponding SPHs.
Figure 8: Effect of foam stabilizer type on water absorbency of GG-SPH, LSD at 5% degrees of
freedom 1.24, CV is 3.246 and F value between is 250.45. Synthesis parameters: weight ratio of monomer, cross
linker, initiator, foam stabilizer, porogen to backbone: 1.5:1, 0.01:1, 0.05:1, 0.1:1 and 0.1: 1, respectively, V H₂O
5.95 ml/ g feed.
Concentration of the porogen influences the swelling properties of the hydrogels. As
is clear from the Table 1, under all the experiment conditions employed in the present study
porogen concentration in the range of 0.07-1.2%, resulted in the SPHs with maximum WAC.
At higher concentrations, consistently low swelling behaviour was exhibited by the SPHs.
Under acidic pH conditions employed (pH 5 to 5.5), SBC reacts with acid to generate CO₂
gas bubbles. Beyond an optimum concentration of the porogen, the pH of the reaction
medium tends to rise which adversely affects the gelation process. This effect is reflected in
inferior water absorbency values of the resulting SPH. Similar observation on the
performance of pH-sensitive SPHs has been reported earlier (Chen et al., 2000).
As is evident, water serves as the medium for gelation and foaming processes to occur
smoothly. Its quantity per unit feed mass was studied as a variable against the swelling
capacity QH₂O of the prepared SPH. The results presented in Table 3 indicated the significant
increase in QH₂O with increase in water volume up to 5.95 ml/g feed. On further increase
60
gelation failed to occur and SPHs were not formed. An optimum dilution leads to effective
collisions of the monomer and cross linker free radicals with the grafting sites of the back
bone, leading to increase in gelation rate and effective entrapment of gas bubbles in the
expanding gel. At higher dilutions, namely low monomer concentration and availability of
more molecular oxygen in the reaction mixture play adverse role and the gel fails to form.
In previous reports on SPHs (Kuang et al., 2011; Chen and Park, 2000; Chavda et al.,
2010), acetic acid or acrylic acid were most commonly employed foaming aids. In our efforts
to find better alternative to these toxic reagents, three organic acids were evaluated for their
pore inducing ability. The results are shown in Figure 9. SPH prepared by using OA-3, a
polycarboxylic acid exhibited most superior water absorbency value, as compared to OA-1
(acetic acid) and OA-2 (a polyfunctional carboxylic acid). Release of higher number of moles
of CO2 in the presence of OA-3 as compared to OA-1 and OA-2 at a particular concentration
of sodium bicarbonate (SBC) may explain the observed behaviour. A comparison with
hydrogel prepared in the absence of the foaming aid (QH₂O 22.7 g/g) justifies the role of
foaming aid in influencing the swelling characteristics carbons of hydrogels. In the absence
of CO2 source, pore formation does not take place in the hydrogel, as confirmed by its SEM
imaging also (Figure 4).
Figure 9: Effect of foaming aid type on water absorbency of GG-SPH, LSD at 5% degrees of
freedom is 1.90, CV is 1.30 and F value between treatments is 1524.13, Synthesis parameters: weight ratio of
monomer, cross linker, initiator, foam stabilizer, porogen to backbone: 1.5:1, 0.01: 1, 0.05:1, 0.1:1 and 0.1: 1,
respectively, V H₂O 5.95 ml/ g feed.
In biopolymer based porous hydrogels, backbone-monomer ratio significantly
influences the swelling properties of the finished product. In most of the studies reported so
far (Chen et al., 2003; Yin et al., 2007) the backbone-monomer ratio was kept very low
61
(<0.2). Synthesis in these studies was intended to generate extremely fast swelling porous
hydrogels for their use as gastric retention release devices, where fast delivery of the inputs is
desirable. In the present study, carrier properties of porous hydrogels to entrap bioagents or
agriculturally important microbes are of interest. Stability requirements over a range of
temperatures coupled with biocompatibility of carrier with the microbes necessitated
presence of higher biopolymer content in the finished products. The effect of backbone-
monomer ratio on the swelling characteristic carbons of the SPHs is presented in Figure 10. It
is clear from the figure that higher backbone content in the feed decreased the swelling
potential of the product. This can be ascribed to the fact that higher concentration of
polysaccharide leads to the increase in the viscosity of reaction medium and also restricts the
disentanglement of polymer chains.
Figure 10: Effect of backbone : monomer ratio on water absorbency of GG-SPH, LSD at 5%
degrees of freedom 1.19, CV is 1.53 and F value between 975.31. Synthesis parameters: weight ratio of
monomer, cross linker, initiator, foam stabilizer and porogen tobackbone:1.5: 1, 0.01:1, 0.05:1, 0.1:1 and 0.1: 1,
respectively, V H₂O 5.95 ml/ g feed.
5.3.3 Effect of duration of reaction on water absorbency
The gelation and foaming processes in the hydrogels prepared in present study were achieved
in less than 2 minutes. However, in order to obtain a stable network, the graft polymerization
reaction was allowed to continue for different time periods.
62
Figure 11: Effect of duration of reaction on water absorbency of GG-SPH, LSD at 5% degrees
of freedom 1.19, CV is 1.53 and F value between 975.31. Synthesis parameters: weight ratio of monomer, cross
linker, initiator, foam stabilizer and porogen to backbone: 1.5: 1, 0.01:1, 0.05:1, 0.1:1 and 0.1: 1, respectively,
VH₂O 5.95 ml/ g feed.
SPHs generated were evaluated for their swelling behaviour in distilled water. The
results are shown in Figure 11. The 6 hr reaction yielded SPH with highest QH₂O (52.3 g/g).
Reaction time more than 6 hours resulted in decrease in water absorbency. This can be
attributed to increase in the cross-link density and screening of pores by the polymer network
chains, when polymerization is allowed after an optimum time period.
5.3.4 Swelling rate
Comparative variation of QH₂O of SPH vs. corresponding porogen free SAP with time in
distilled water at 50⁰ C is shown in Figure 12. It is evident the SPH swells fast attaining 50%
absorbency in 30 minutes. After 30 minutes, swelling ratio consistently increased and
attained its maximum equilibrium swelling in 6 hr. The porogen free SAP, on the other hand
reached its QH₂O maximum in 24 hr. Initial fast swelling in case og GG-SPH is due to
capillary action through fast action through the pores. Later on, macromolecular expansion
seems to influence the swelling rate. In case of SAP, the swelling occurs at much slower rate
and is diffusion controlled.
5.3.5 Effect of external environment on water absorbency
The effect of temperature of the swelling medium i.e., on the water absorbency of SPH is
shown in Figure 13.
63
Figure 12: Effect of time period on water absorbency of GG-SPH vs. GG-SAP
LSD at 5% degree of freedom iss 4.33, CV is 4.09 and F value is 1436.1
Figure 13: Effect of temperature of swelling medium on water absorbency of GG-SPH
Rise in temperature of swelling medium exhibited increase in QH₂O. Maximum
swelling is observed at 50⁰ C. This behaviour can be due to the extensive entanglement or
expansion of the macromolecular chains at higher temperatures.
The swelling behaviour of SPH in different pH environments is presented in Table 5.
At pH 9, the hydrogel shows maximum swelling. This can be explained in terms of ionization
of the amide and -COOH groups to COO-
groups. The electrostatic repulsion between the
64
COO- groups leads to macromolecular expansion and the resulting enhancement of QH₂O. In
acidic pH, protonation of the -CONH2 and –COO-
groups leads to decrease in extent of
hydrogen bonding with water molecules and thus the decrease in water absorbency. Similarly
findings have been confirmed in previous studies on SPHs and SAPs (Singh et al., 2010).
Swelling behaviour of the SPH in different salt solutions namely ammonium sulphate
(AS), ammonium nitrate (AN), potassium nitrate (PN), sodium chloride (SC) and fertilizer
urea solution, of strengths 5 mM, 10 mM, 15 mM and 20 mM is depicted in Table 5. It is
clear from the Table 3, that the hydrogel shows drastic reduction in its swelling tendency in
all the salt solutions. Similar observation has been reported by Gils et al., 2009. The cations
in the salt solutions screen the polar groups causing reduction in the hydrogen bonding with
H₂O molecules. Also, in the salt solutions, the osmotic pressure resulting from the difference
in mobile ion concentration between the gel matrix and the surrounding aqueous phase
decreases, adversely affecting absorbency. In urea solution also, the absorbency exhibited fall
with increase in its concentration from 5 mM to 20 mM, though the overall reduction in QH₂O
was lesser than that in salt solutions.
In our previous work (Singh et al., 2010) we have reported that water quality also
influences the swelling performance of hydrogels. The effect of water quality on the swelling
behaviour of SPH is shown in Table 4. As compared to deionized water, absorbency is
significantly reduced in different aqueous environments. The observed behaviour may be
attributed to the reduction in osmotic potential between gel and the surrounding water
containing cations of varying strengths (Pourjavadi et al., 2008).
5.3.6 Porosity and density measurements
Effect of variation in cross linker concentration on the density and porosity of the cl-guar
gum-g-PAam SPHs is presented in Table 6. The results shown here offer an interesting
pattern in contrast to previous studies reported on SPHs. Density of the SPHs increased with
the increase in cross linker concentration whereas porosity varied inversely. Conventionally
as described by Kabiri et al., (2003), higher cross linker concentrations should lead to shorter
gelation times due to which the CO2 gas bubbles cannot escape from the reaction mixture and
thus the porosity increases. Similarly, due to generation of high foam volume, density of the
hydrogel decreased with increase in cross linker concentration. The ironical findings in the
present study can be understood as follows. For the network formation of optimum
65
Table 3: Effect of various synthesis parameters on water absorbency of GG-SPH
Cross linker
concentration1
(wt %)
Water
absorption
Q H₂O (g/g)
Foam
stabilizer
concentration2
(wt%)
Water
absorption
Q H₂O
(g/g)
Water
volume3
(ml/g)
Water
absorption
Q H₂O (g/g)
Porogen
concentration4
(wt %)
Water
absorption
Q H₂O (g/g)
0.01 11.2c
0.0 22.7e
1.98 26.3e
0.1-0.5 32.9g
0.03 16.8bc
0.3 25.6d
3.96 37.8c
00.7-1.2 48.3a
0.1 20.3b
1.8 35.9c
5.95 48.3a
1.4-2.2 46.36b
0.3 45.3a
3.6 43.5a
7.93 39.7b
2.3-2.9 44.9c
0.7 47.2a
7.3 40.6b
9.92 33.1d
3.1-4.4 40.9d
4.8-6.6 38.2e
6.9-9.1 35f
LSD at 5% 6.18 1.20 1.47 0.91
CV 11.65 1.89 2.11 1.25
F value 79.01 617.94 325.13 393.39
Means within a column followed by different letters are significantly different at 5% level of significance and those following by the same letter do not significantly at 5%
level of significance. 1Synthesis parameters: weight ratio of monomer, initiator, foam stabilizer, porogen to backbone: 1.5:1, 0.05:1, 0.1:1 and 0.1: 1, respectively, V H₂O 5.95 ml/ g feed.
2Synthesis
parameters: weight ratio of monomer, cross linker, initiator, porogen to backbone: 1.5:1, 0.01: 1, 0.05:1 and 0.1: 1, respectively, V H₂O 5.95 ml/ g feed. 3Synthesis parameters:
weight ratio of monomer, cross linker, initiator, foam stabilizer, porogen to backbone: 1.5: 1, 0.01:1, 0.05:1, 0.1:1 and 0.1: 1, respectively. 4Synthesis parameters: weight
ratio of monomer, cross linker, initiator and foam stabilizer to backbone: 1.5:1, 0.01: 1, 0.05:1 and 0.1: 1, respectively, V H₂O 5.95 ml/ g feed
66
Table 4: Effect of pH and quality of water on water absorbency of GG-SPH
pH of water Water absorption
Q H₂O (g/g)
Quality of water Water absorption
Q H₂O (g/g)
Acid (4) 43.1c
Deionized water 196.8a
Neutral (7) 164.5b
Distilled water 166.2b
Alkaline (9) 199.6a
Tap water 114.6c
Hard water -A 85.9d
Hard water –B 61.8e
Hard water -C 38.7f
LSD at 5% 4.44 5.40
CV 1.44 2.68
F value 5250.30 1270.81
Means within a column followed by different letters are significantly different at 5% level of significance and
those following by the same letter do not significantly at 5% level of significance.
Table 5: Effect of salt/ fertilizer type and their strength on water absorbency of GG-SPH
Water absorption at different salt concentration (Q H₂O (g/g))
Salt/Fertilizer type 5 mM 10 mM 15 mM 20 mM
(NH4)SO4 51.83h
34.5kl
21.4mn
19.2n
NH4NO3 62.6g
42.8j
40.6j
32.5l
KNO3 68.5f
51.8h
45.9i
40.9j
NaCl 71.6e
51.4h
36.2k
23.2m
NH2CONH2 133.4a
110.6b
94.1c
76.7d
LSD at 5% 2.41
CV 2.62
F value 1269.57
Means within a column followed by different letters are significantly different at 5% level of significance and
those following by the same letter do not differ significantly at 5% level of significance.
crosslinking density, a threshold quantity of cross linker is necessary which is specific to the
experimental conditions, back bone nature and content etc. The minimum cross linker
concentration screened in the present study was in the range of 0.005-0.02 wt % and the
maximum was 0.7-0.8 wt %. The screened concentration range is much lower than that
reported previously (Chavda et al., 2010; Kabiri et al., 2003). Low crosslinker content may
not be just sufficient to generate a network through grafting and cross linking. Therefore an
67
increase in the density and decrease in porosity is observed with increase in concentration of
the cross linker. Findings from the present study will be utilised in our future endeavours, to
develop cost effective process for production of guar gum based porous hydrogels.
Table 6: Effect of cross linker concentration on density and porosity on SPHs
Cross linker concentration
(g)
Density (g/cc) Porosity (%)
0.0005 0.61e
79.9a
0.001 0.67d
70.7b
0.005 0.71c
63.2c
0.01 0.79b
58.4d
0.02 0.86a
32.8e
LSD at 5% 0.012 2.20
CV 0.92 1.92
F value 603.39 686.95
Means within a column followed by different letters are significantly different at 5% level of significance
(p<0.0001) and those following by the same letter do not significantly at 5% level of significance.
5.4 Conclusions
Novel cl-gg-g-PAam superporous hydrogels were prepared employing a novel process of
preparation. Effect of reaction parameters and reagents on the swelling behaviour of SPHs
was investigated. Variation of concentration of the cross linker led to hitherto unreported
observations regarding its effect on the swelling behaviour, porosity and density of dry SPH.
SEM analysis of the hydrogel revealed porous structure. This feature coupled with the
biocompatible character of guar gum backbone indicates potential of these SPHs as carriers
of agro-inputs particularly agriculturally important microbes. In our future endeavour, the
SPH will be explored as carrier materials to entrap microbes with optimized swelling
characteristics.
68
RESEARCH PAPER III
.
Novel bioformulations of Trichoderma harzianum and Pseudomonas fluorescens: shelf
life characteristics and bioefficacy against Pythium aphanidermatum (in vitro)
Abstract
Pythium aphanidermatum, a soil borne fungal pathogen, causes extensive damage in number
of crops. It is causative factor of root rot disease in vegetable and fruit crops. Control of
Pythium primarily involves application of chemical fungicides Use of soil borne non-
pathogenic microorganisms of the genera Trichoderma and Pseudomonas represents an eco-
friendly alternative to the chemical approach. The present study was conducted to assess
viability and bioefficacy (in vitro) of composite formulations of Trichoderma harzianum
(Thz) and Pseudomonas floresecence (Pflo) prepared by, SAP and SPH as carriers. Thz and
Pflo exhibited 68% and 88% respective growths on Petri plate in presence of each other. The
shelf-life evaluation in terms of viability of bio-agent spores/cells as a function of
temperature (5, 25 and 45 ⁰C) and time period (0, 15, 30, 60, 90, 120, 150, 180 days) was
evaluated. Both SAP and SPH compositions exhibited significantly high viability of Thz and
Pflo (>108 cfu/g carrier) till the last day of study period. All the combination formulations
(Thz and Pflo together), irrespective of the type of carrier and method of preparation
exhibited >50% Pythium inhibition at all the three storage periods. At 450 C storage
temperature, reduction in the percentage inhibition (<50%) was observed in all the
formulations except WSAP-C (53.6%). However, at 5 month storage period, all the
combinations exhibited 51.4 % - 61.5 % inhibition of P. aphanidermatum.
Key words: Shelf-life, hydrogel, bioformulation, bioefficacy
6.1 Introduction
In present era of commercial and high value agriculture, horticultural crops are front runners
for betterment of small and marginal farmers. India is second largest producer of fruits and
vegetables in the world. Unfortunately, many crops suffer from several fungal and viral
diseases. Amongst the fungal diseases, the most serious one is color or root rot disease. It is
caused by Pythium aphanidermatum and Phytopthora parasitica. Pythium aphanidermatum
is an important fungal pathogen which infects plants and causes high mortality of seedlings
grown in the nursery and field (Kannan & Jayaraj, 1998). In general, the pathogen is
69
especially important in high value horticultural crops particularly those produced in
greenhouses and soilless culture. Since the pathogen is soil-borne, several attempts have been
made to control the disease at soil level in several ways. Chemical fungicides such as
Metalaxyl, Azoxystrobin and Pyralclostrobin have been the main weapons in controlling soil-
borne plant pathogens and in increasing the yields in modern systems of crop production
(Ulrike and Werner, 1998; Salman, 2010). Although fungicides are used extensively, the
results are temporary and require frequent application (2-3 times). In view of the
environmental implications of the use of synthetics, alternative strategies for the control of
plant disease are being sought (Weller, 1988; Ellis et al., 1999). Biological control using
antagonistic nonpathogenic microbes alone, or as supplements to minimize the use of
chemical pesticides has become an important component in recent years (Mao et al., 1997;
Hwang, 1994; Emmert & Handelsman, 1999). Biological control of plant diseases using
antagonistic bacteria and fungi offers a powerful and eco-friendly alternative to the use of
synthetic chemicals). Various strains of non-pathogenic fungus T. harzianum and the
bacterium P. fluorescens have been established to control number of pathogens such as
different species of Pythium, Fusarium, Alternaria, Rhizoctonia etc (Verma et al., 2007).
Most of the reported studies on disease biocontrol deal with single biocontrol agent as
antagonist to a single or multiple pathogens (Leemanceau & Alabouvette, 1991). Single
biocontrol agent is not likely to be active in all soil environments. Naturally occurring
biocontrol results from combined action of antagonists rather than from a high population of
a single antagonist. A mixture of antagonists is considered to account for protection in
disease suppressive soils (Hornby, 1983; Lemanceau and Alabouvette, 1991; Chaube et al.,
2003).Strategy of combining two or more antagonists to enhance the level of disease
management is thus imperative. Application of a mixture of introduced antagonists would
more closely mimic the natural situation and enhance the efficacy and reliability of control.
(Janisiewicz, 1988; Duffy and Weller, 1995; Varshney and Chaube, 2001).The activity and
efficacy of bio control agents are often profoundly affected by several factors such as
environmental conditions in the soil and rhizosphere (Dunne et al., 1998; Deacon and Berry
1993), type and amount of inoculum applied, method and timing of application and age of the
inoculi etc. (Lewis and Papavizas 1987a,b). Serious bottlenecks are high temperature and
limited moisture in the soil (Rini et al., 2006). For field application, high inoculation rates of
the desired organism too become essential for satisfactory performance (Nakkeran et al.,
2005). To achieve this, formulation of bio agents in various carriers have been reported
70
(Taweil et al., 2010). One of the most recent technologies for the formulation of biocontrol
agents is the immobilization of wet or dry biomass within cross-linked polymers such as
alginate and carrageenan (Lumsden et al., 1995). Incorporation of fungal mycelia in alginate
pellets has been found successful for the delivery of biocontrol fungi (Knudsen et al., 1990
a,b; Lewis and Papavizas 1985; Papavizas et al., 1987). Polymers, synthetic as well as
natural, have been suggested as potential carriers of microbes (Dommergues et al., 1979;
Jung et al., 1982). In most of the available reports on bioformulations, the viability and shelf
life evaluations have been conducted at 10-200
C storage temperature. In view of high
temperature conditions of tropical and subtropical areas of the world, significant bio agent
survival and viability at higher temperatures can play an important role in their success. In
this context, work has been reported from our laboratory on improving the shelf life
characteristics of microbes under high temperature conditions using hydrogels as carriers
(Mondal, 2012). Hydrogels are a speciality class of hydrophilic polymers known for their
versatile matrix properties (Singh et al., 2010). In order to explore the possibility of
integrating biocontrol potentials of Trichoderma harzianum(Thz) and Pseudomonas
fluorescens(Pflo), two novel hydrogel carriers of semi synthetic nature (SAP and SPH )
developed in our laboratory (water absorption capacity, >600-700 g/g and >160 g/g
respectively at 500C) were employed to prepare twelve formulations of Thz and Pflo (each
alone and in combination). The aim of the present work was to evaluate the shelf life (in
vitro) of the prepared formulations at different storage temperatures and to assess their
effective viability duration. In the second part of the study, bioefficacy of the formulations
stored under different storage temperatures and periods was investigated and has been
presented.
6.2 Materials and Methods
6.2.1 Hydrogel carriers
The biopolymeric hydrogels SAP and SPH employed in the present study were prepared in
our laboratory by free radical graft polymerization technique. Their water absorption capacity
was >600-700 g/g and >160 g/g respectively at 500C. Each dry hydrogel was milled
separately and sieved to obtain particles of size 100-240 mesh. The dry fine powder was
autoclaved (1210C, 15psi, 15 minutes) and used.
71
6.2.2 Plant pathogenic fungus and biocontrol agents
Fresh live cultures of Pythium aphanidermatum, Trichoderma harzianum and Pseudomonas
fluorescens were obtained from ITCC (Indian Type Culture Collection) section of Plant
Pathology Division, Indian Agricultural Research Institute, New Delhi, India. The fungi
cultures were maintained on Potato Dextrose Agar slants in BOD incubator at 28 ± 2°C for
multiplication. The bacterium culture was maintained on Nutrient Agar slants in BOD
incubator at 28 ± 2°C. All were sub-cultured in test tubes and Petri plates prior to testing.
6.2.3 Microbial growth media
For fungal growth, analytical grade Potato Dextrose Agar (PDA) media was purchased from
Merck Specialities Pvt. Ltd., Mumbai, India (pH 5.6 ± 0.2 at 250C). Analytical grade Potato
Dextrose Broth (PDB) media was purchased from Hi-media Laboratories Pvt. Ltd., Mumbai,
India (pH 5.1 ± 0.2 at 250C). For bacterial growth, analytical grade Nutrient Agar (NA)
media was purchased from Merck Specialities Pvt. Ltd., Mumbai, India (pH 5.6 ± 0.2 at
250C). Analytical grade Nutrient Broth (NB) media was purchased from Hi-media
Laboratories Pvt. Ltd., Mumbai, India (pH 5.1 ± 0.2 at 250C).
6.2.5 In vitro compatibility evaluation of Pflo and Thz and their antagonistic activity
against Pythium aphanidermatum
The compatibility between Pflo and Thz was evaluated in vitro by poisoned food technique
(Siddique et al., 2009) using Potato Dextrose Agar (PDA) medium and nutrient agar (NA)
medium. For fungi, PDA suspension in water was boiled to obtain uniform media. Similarly
for bacteria, NA madia was prepared. For combinations (bacteria & fungi), mixture of NA
and PDA (3:7) was used. The media (50 ml) was transferred to conical flasks of 100 ml
capacity The media and Petri plates were put in sterilized autoclaved bags and autoclaved at
15 psi for half an hour prior to use. PDB media was prepared by dissolving 24 g PDB in 1000
mL distilled water. NB media was prepared by dissolving 13 g NB in 1000 mL distilled
water. Same precautions were taken as in case of PDA media.
Sterilized mixture of NA and PDA media (3:7) was poured into 92 mm diameter Petri
plates @ 20 ml per plate and 5 mm diameter solid discs of inoculum of each of the bacterium
and fungus was placed near the edges of the Petri plates. Individual inoculum was placed in
72
the centre of dish separately as control. Three replicates were taken for each treatment. Petri
plates were incubated in BOD incubator @ 28oC. Growth of each organism was recorded on
when the upper surface of solidified Petri plate was just full with microbial growth as in case
of control. Percent compatibility was calculated as: % compatibility = 100 - % inhibition.
For antagonistic study of bioagents against Pytium aphanidermatum, mycelia agar
plug of the pathogen was placed in the centre and biocontrol agent was kept at periphery of
Petri plate for individual bioassay. For evaluation of joint activity, of Trichoderma harzianum
and Pseudomonas fluorescens, the pathogen was placed at centre and both bioagents were
placed separately on either side of Petri plates. Pythium aphanidermatum was used as control.
Percent growth inhibition was calculated as: I = (C – T)/C × 100, where I = Percent growth
inhibition, C = Colony diameter in control and T = Colony diameter in treatment
6.2.6 Preparation of bioformulations of Trichoderma harzianum and Pseudomonas
fluorescens
Hydrogels, SAP and SPH were used as carriers to develop formulation containing Thz and
Pflo (alone and in combinations). Details of the complete process will be protected under
IPR. Briefly, two types of formulations were prepared, dry (moisture 0-5%) and wet
(moisture 10-60%). Precaution was taken to introduce (108-10
11) colony forming units of
bioagents per gram carrier in all compositions. Twelve compositions (Table 1) were prepared
and preserved at three temperatures (5⁰, 25⁰ and 45⁰ C) in BOD incubators for shelflife
evaluation.
Table 1: Compositions used in the bioefficacy study against Pythium aphanidermatum
Sl.No. Composition
1. Wet formulation of Thz in SAP (WSAP-Thz)
2. Wet formulation of Pflo in SAP (WSAP-Pflo)
3. Wet formulation of Thz and Pflo mixture in SAP(WSAP-C)
4. Dry formulation of Thz in SAP (DSAP-Thz)
5. Dry formulation of Pflo in SAP (DSAP-Pflo)
6. Dry formulation of Thz and Pflo mixture in SAP (DSAP-C)
7. Wet formulation of Thz in SPH (WSPH-Thz)
8. Wet formulation of Pflo in SPH (WSPH-C)
9. Wet formulation of Thz and Pflo mixture in SPH (WSPH-C)
10. Dry formulation of Thz in SPH (DSPH-Thz)
11. Dry formulation of Pflo in SPH (DSPH-Pflo)
12. Dry formulation of Thz and Pflo mixture in SPH (DSPH-C)
73
6.2.7 Shelf life study
All the twelve compositions and the absolute controls of Thz and Pflo were kept at three
storage temperatures 5, 25 and 45 0C in B.O.D. incubators. Sampling was done periodically
at intervals of 0, 15, 30, 60, 90, 120, 150 and 180 days. Percent viability in terms of log cfus
per gram formulation was calculated.
6.2.8 Bioefficacy study (in vitro) against Pythium aphanidermatum
All the twelve test compositions and three controls namely Thz, Pflo and their mixture (1:1),
stored at three storage temperatures i.e., 5⁰, 25⁰ and 45⁰ C were evaluated periodically (0 day,
15 day, 30 day, 60 day. 90 day, 120 day, 150 day and 180 day) for their bioefficacy against
Pythium aphanidermatum (in vitro). Test composition (0.1 g) from each treatment was
uniformly dispersed in the growth medium in petri plate. Petri plates were kept at B.O.D.
incubator at 280C. After 5-7 days, when the growth of pathogen became maximum inhibition
% was calculated from the reduction of diameter in treated Petri plates. Percent inhibition
was calculated as ibid.
6.2.9 Statistical analysis
Statistical analysis of all the laboratory CRD experiments was done using PROC GLM SAS
(9.2) of SAS Institute, Cary, NC.
6.3 Results and Discussions
6.3.1 Effect of storage temperatures on viability of fungus and bacteria immobilized in
various formulations
Viability behaviour of microbes in all the test compositions including controls is presented in
Table 3(a-c). Relative reduction in number of viable units of Thz and Pflo expressed here as
log c.f.u g-1
is presented in Figure 2. It is evident from the Tables that Thz and Pflo
formulated singly or as mixture in dry and wet formulations sustained significantly high
viability (>108 c.f.us/g) at all the storage temperatures. This can be attributed to the
hydrophilic and network character of hydrogel carriers. SEM and compound microscopic
74
Figure 1: SEM Images of SAP (A) and SPH (B) carriers; SAP bioformulations of Thz (C and
D); SPH bioformulation of Thz (E); SAP bioformulation of Pflo (F); Compound microscopic
images of Pflo(G) and Thz (H).
Thz spores
Pflo cells
A
H G
F E
D C
B
Thz spores
Thz spores
Thz spores
Pflo cells
75
images of the bioformulations (Fig. 1) depict extensive entrapment and distribution of Thz
spores and mycelia and Pflo cells in the hydrogel network.
Observed viability reported herein particularly at 450 C is hitherto unknown in
literature. Bazilah et al. (2011) reported that vermiculite served better than coir pith and
maximum viability was observed at lower temperatures.
Moisture availability around the microbial units was cited as a major attribute
(Beatrice et al., 1991; Cigdem and Merin, 2005; Walker et al., 2004). Nakkeran in 2005
reported that higher water holding capacity is an essential characteristic of a good carrier.
This observation is also confirmed by other workers (Weiss et al., 1987; Elazar et al., 1966;
Loccoz, 1999). Formulations evaluated in the present study are based on high water
absorbing biopolymer hydrogels. Even though the moisture content of the dry formulations
was much less than the wet formulations, owing to their hydrophilic nature, on addition to the
agar media, the hydrogels tended to absorb moisture of the growth media, releasing cells/
spores which revived. At all the storage temperatures, the four combination formulations i.e.,
WSAPC, WSPHC, DSAPC and DSPHC exhibited reduction in the viability of Thz and Pflo
as compared to their respective individual formulations. This could be due to the competition
between the two antagonistic microbes and the results of in vitro compatibility study
confirmed the same. Amongst combination formulations, the viability reduction is lesser in
SPH based compositions. This is likely due to extensive porosity in SPH as compared to the
SAP carrier. Presence of channels in the matrix of SPH may be serving as the niches for the
microbial cells/ spores and thus, due to less crowding, the percent viability of the individual
microbes in the WSPHC and DSPHC showed lesser decline than their SAP counterparts. As
seen in the Fig 2, percent reduction in viable cell count over six months in all the
formulations showed an increase with temperature. On 180 day, viability of test formulations
as function of storage temperature is shown in Table 2. It is evident that except WSAP-Pflo,
all the test formulations exhibited decline in viable cell count with increase in temperature.
Difference was more between viability 25⁰ and 45⁰ C. Overall WSAPC suffered maximum
fall in viability of both Thz and Pflo.
6.3.2 Bioefficacy of different formulations vis a vis controls
All the combination formulations, irrespective of the type of carrier and method of
preparation exhibited >50% Pythium inhibition at all the three storage periods. At 50C and 6
76
months storage time, while control treatments comprising Thz, Pflo and their mixture showed
28-45% inhibition of Pythium, all the test formulations exhibited percentage inhibition in the
range of 52% to 76%. Similarly at 250C, a very low percentage inhibition (19-28%) could be
achieved in controls, whereas all the test formulations exhibited significantly higher
bioefficacy. In particular, WSAP formulations of individual bioagents as well as their mixture
performed best with 53-66% reduction in the pathogen growth. It is clear from Tables (3a, 3b
and 3c) that although the bioefficacy of formulations exhibited gradual decline particularly
after 4 months of storage at 450C, as compared to absolute controls, a very superior
performance by all the formulations could be achieved.
Table 2: Viability behaviour of test compositions on 180th
day at different storage
temperatures
180 day Log CFUs
Type Formulations 5° C 25° C 45° C LSD
Wet WSAP-Thz 10.5a
10.1b
9.8c 0.291
WSAP-Pflo 10.4a
10.3a
9.9a 0.529
WSAPC-Thz 9.8a
9.6b
9.0d 0.140
WSAPC-Pflo 9.9a
9.8a
9.2c
WSPH-Thz 9.8a
9.7a
9.5b 0.218
WSPH-Pflo 10.2a
9.9b
9.6b 0.301
WSPHC-Thz 9.8c
9.8bc
8.8e 0.157
WSPHC-Pflo 10.0a
9.2ab
9.2d
Dry DSAP-Thz 10.1a
9.9ab
9.9b 0.132
DSAP-Pflo 10.3a
9.9b
9.8c 0.151
DSAPC-Thz 9.9b
9.7c
8.9e 0.165
DSAPC-Pflo 10.0a
10.0ab
9.2d
DSPH-Thz 10.0a
9.8ab
9.7b 0.167
DSPH-Pflo 10.2a
9.9b
9.7c 0.119
DSPHC-Thz 9.9b
9.9b
9.5d 0.101
DSPHC-Pflo 10.0a
10.0ab
9.7c
Control Thz 0.5a
0.5a
0.4a 0.113
Pflo 0.6a
0.6a
0.5a 0.084
C-Thz 0.7c
0.7c
0.7bc 0.054
C-Pflo 0.8a
0.7ab
0.8a
Means within a row followed by different letters are significantly different at 5% level of significance
and those following by the same letter do not significantly at 5% level of significance
The percentage inhibition caused by combination formulations WSAPC, WSPHC and
DSPHC at all temperatures across 180 days was significantly higher as compared to the
corresponding carrier based formulation of individual Thz and Pflo. A gradual reduction in
the observed bioefficacy of all the test formulations was observed with rise in storage
77
temperature (Table 4a, 4b and 4c). As seen earlier in Table 2, significantly high viability of
microbes was observed in all test compositions from which consistently higher bioefficacy
behaviour of the prepared compositions was expected. On the contrary, observed reduction at
higher temperatures and longer storage durations can be attributed to the method and quantity
of application of formulation in in vitro bioefficacy studies. This aspect will be in our future
studies. In general, the wet formulations of Thz or Pflo or mixture showed consistently higher
percent inhibition of Pythium.
6.4 Conclusion
Novel hydrogel based bioformulations of Trichoderma harzianum and Pseudomonas
fluorescens were evaluated in terms of their extended shelf life and bioefficacy against
Pythium aphanidermatum in vitro. All the test formulations exhibited significant viability (>
108 c.f.u. per gram) even after 6 months at all the storage temperatures. Behaviour at 45
0C is
heitherto unknown in the literature. All the combination formulations exhibited superior
bioefficay against Pythium for 5 months at all the storage temperatures. The lower observed
bioefficacy during long term storage period can be attributed to the method and the amount of
application of the developed formulations. Findings from the present study point towards
potential bioformulations that possess superior shelf life characteristics as compared to those
reported in literature. Still, their actual potential needs be evaluated under greenhouse and
field conditions.
78
Table 3a: Shelf life of bioagents in formulations with control at 5⁰ C storage temperature
Log CFUs
Type Formulations 0 day 15 day 30 day 60 day 90 day 120 day 150 day 180 day LSD
Wet WSAP-Thz
11.0a
11.0ab
10.9bc
10.9c 10.8
cd 10.8
de 10.7
e 10.5
f 0.116
WSAP-Pflo
11.1a
11.0ab
11.0ab
10.9abc
10.8bc
10.8dc
10.6d
10.4e
0.208
WSAPC-Thz 10.2a
10.2a
10.2ab
10.1bc
10.0cd
9.9de
9.9e
9.8f
0.079
WSAPC- Pflo 10.3a
10.2a
10.2ab
10.2bc
10.1c 10.0
d 10.0
d 9.9
e 0.068
WSPH-Thz 10.9a
10.8bc
10.7c 10.6
d 10.4
e 10.1
e 10.0
e 9.8
f 0.147
WSPH-Pflo 11.0a
11.0ab
10.9ab
10.9ab
10.8bc
10.8c 10.2
d 10.2
d 0.127
WSPHC-Thz 10.2a
10.1ab
10.1ab
10.1b 10.0
c 10.0
cd 9.9
d 9.8
e 0.080
WSPHC-Pflo 10.3a
10.2ab
10.2bc
10.2bc
10.2cd
10.1d 10.0
e 10.0
e 0.053
Dry DSAP-Thz 11.0a
10.9a
10.8ab
10.8b 10.5
c 10.4
c 10.2
d 10.1
e 0.144
DSAP-Pflo 11.1a
11.0b
10.9bc
10.9cd
10.8d 10.8
e 10.6
f 10.3
g 0.074
DSAPC-Thz 10.2a
10.2ab
10.2ab
10.2bc
10.1c 10.0
d 10.0
d 9.9
e 0.079
DSAPC-Pflo 10.3a
10.3a
10.3ab
10.2bc
10.2dc
10.2d 10.1
e 10.0
f 0.048
DSPH-Thz 11.0a
10.9a
10.8b 10.7
b 10.6
c 10.4
d 10.2
e 10.0
f 0.099
DSPH-Pflo 11.0a
11.0a
11.0a 10.9
a 10.9
a 10.8
b 10.4
c 10.2
c 0.112
DSPHC-Thz 10.2a
10.2a
10.1ab
10.1bc
10.1c 10.0
d 10.0
d 9.9
e 0.056
DSPHC-Pflo 10.3a
10.2a
10.2ab
10.2bc
10.2c 10.2
c 10.1
d 10.0
e 0.042
Control Thz 11.0a
10.0b
8.0c
6.5d
4.7e
2.2f
0.9g
0.5h
0.068
Pflo 11.1a
10.1b
8.0c
6.6d
4.8e
2.3f
1.0g
0.6h
0.101
C-Thz 10.2a
9.2b
7.1c
5.7d
3.9e
1.4f
1.1g
0.7h
0.104
C-Pflo 10.3a
9.3b
7.3c
5.7d
4.0e
1.5f
1.2g
0.8h
0.041
Means within a row followed by different letters are significantly different at 5% level of significance and those following by the same letter do not significantly at 5% level
of significance.
79
Table 3b: Shelf life of bioagents in formulations with control at 25⁰ C storage temperature
Log CFUs
Type Formulations 0 day 15 day 30 day 60 day 90 day 120 day 150 day 180 day LSD
Wet WSAP-Thz 11.0a
11.0a
10.9ab
10.8bc
10.7cd
10.6d 10.4
e 10.1
f 0.128
WSAP-Pflo 11.1a
11.0ab
10.9abc
10.9abc
10.8bc
10.7cd
10.5d
10.3e
0.252
WSAPC-Thz 10.2a
10.2a
10.1ab
10.0bc
10.0cd
9.9de
9.8e
9.6f
0.091
WSAPC-Pflo 10.3a
10.2a
10.2ab
10.1b 10.1
b 10.0
d 9.9
d 9.8
e 0.066
WSPH-Thz 10.9a
10.8b
10.6c 10.6
c 10.3
d 10.0
e 9.9
e 9.7
f 0.093
WSPH-Pflo 11.0a
11.0a
10.9b 10.8
bc 10.7
c 10.4
d 10.0
e 9.9
f 0.341
WSPHC-Thz 10.2a
10.1ab
10.1abc
10.1bc
10.0cd
10.0de
9.9e
9.8f
0.082
WSPHC-Pflo 10.3a
10.2a
10.1b 10.0
b 10.0
c 9.9
c 9.9
d 9.2
e 0.046
Dry DSAP-Thz 10.9a
10.9ab
10.8bc
10.7c 10.5
d 10.3
e 10.2
f 9.9
g 0.114
DSAP-Pflo 10.9a
10.9a
10.8b 10.7
bc 10.5
c 10.3
d 10.2
e 9.9
f 0.102
DSAPC-Thz 10.3a
10.2a
10.2ab
10.1bc
10.1dc
10.1de
10.0e
9.7f
0.07
DSAPC-Pflo 10.3a
10.3ab
10.3b 10.2
c 10.2
c 10.2
c 10.1
d 10.0
e 0.04
DSPH-Thz 11.0a
10.8ab
10.7bc
10.7c 10.4
d 10.2
e 10.1
e 9.8
f 0.129
DSPH-Pflo
11.0a
11.0a
10.9b 10.9
b 10.8
c 10.6
d 10.2
e 9.9
f 0.086
DSPHC-Thz 10.2a
10.1ab
10.1ab
10.1bc
10.1cd
10.0d 10.0
e 9.9
e 0.057
DSPHC-Pflo 10.2a
10.2ab
10.2c
10.2c 10.1
d 10.1
e 10.0
e 10.0
f 0.039
Control Thz 11.0a
10.0b
8.0c
6.5d
4.7e
2.2f
0.9g
0.5h
0.122
Pflo 11.1a
10.1b
8.1c
6.6d
4.8e
2.3f
1.0g
0.6h
0.034
C-Thz 10.2a
9.2b
7.1c
5.7d
3.9e
1.4f
1.1g
0.7h
0.029
C-Pflo 10.2a
9.2b
7.2c
5.7d
3.9e
1.4f
1.1g
0.7h
0.043
Means within a row followed by different letters are significantly different at 5% level of significance and those following by the same letter do not significantly at 5% level
of significance.
80
Table 3c: Shelf life of bioagents in formulations with control at 45⁰ C storage temperature
Log CFUs
Type Formulations 0 day 15 day 30 day 60 day 90 day 120 day 150 day 180 day LSD
Wet WSAP-Thz 10.9a
10.9a
10.8ab
10.7bc
10.6cd
10.4d 10.1
e 9.8
f 0.175
WSAP-Pflo 11.0a
11.0ab
10.9bc
10.8c 10.7
d 10.5
e 10.1
f 9.9
g 0.094
WSAPC-Thz 10.2a
10.1a
10.1a 10.0
b 9.9
b 9.7
c 9.6
d 9.0
e 0.087
WSAPC-Pflo 10.2a
10.2a
10.2a 10.0
b 10.0
b 9.9
c 9.7
d 9.2
e 0.082
WSPH-Thz 10.9a
10.7b
10.5c 10.4
c 10.0
d 9.9
d 9.7
e 9.5
f 0.124
WSPH-Pflo 11.0a
10.9ab
10.8bc
10.7c 10.4
d 10.1
e 9.9
e 9.6
f 0.186
WSPHC-Thz 10.2a
10.1ab
10.1b 9.9
c 9.9
c 9.7
d 9.5
e 8.8
f 0.092
WSPHC-Pflo 10.3a
10.2ab
10.1bc
10.0cd
10.0de
9.9e
9.7f
9.2g
0.107
Dry DSAP-Thz 11.0a
10.9a
10.8b 10.6
c 10.3
d 10.1
e 10.0
e 9.9
f 0.112
DSAP-Pflo 11.0a
10.9ab
10.8bc
10.6cd
10.5d 10.4
e 10.1
f 9.8
g 0.138
DSAPC-Thz 10.2a
10.2a
10.2ab
10.1ab
10.0bc
9.9c
9.3d
8.9e
0.167
DSAPC-Pflo 10.3a
10.3a
10.2a 10.2
ab 10.1
bc 10.0
c 9.7
d 9.2
e 0.104
DSPH-Thz 11.0a
10.8b
10.6c 10.4
d 10.2
e 10.0
ef 10.0
f 9.7
g 0.139
DSPH-Pflo 11.0a
11.0a
10.8b 10.8
b 10.6
c 10.2
d 10.2
d 9.7
e 0.119
DSPHC-Thz 10.2a
10.1ab
10.1bc
10.0c 9.9
d 9.9
d 9.7
e 9.5
f 0.066
DSPHC-Pflo 10.3a
10.2ab
10.2bc
10.1c 10.0
d 10.0
de 9.9
e 9.7
f 0.072
Control Thz 10.9a
9.9b
7.9c
6.4d
4.6e
2.1f
0.8g
0.4h
0.096
Pflo 11.0a
10.0b
8.0c
6.5d
4.7e
2.2f
0.9g
0.5h
0.068
C-Thz 10.2a
9.2b
7.2c
5.7d
3.9e
1.4f
1.1g
0.7h
0.028
C-Pflo 10.3a
9.3b
7.3c
5.8d
4.0e
1.5f
1.2g
0.8h
0.023
Means within a row followed by different letters are significantly different at 5% level of significance and those following by the same letter do not significantly at 5% level
of significance.
81
Table 4a: Bioefficacy of formulations with control at 5⁰ C storage temperature
% Inhibition
Type Formulation 0 day 15 day 30 day 60 day 90 day 120 day 150 day 180 day
Wet WSAP-Thz 77.03g 76.3g 75.3g 73.2f 72d 70.2e 64.4e 61.1f
WSAP-Pflo 87.8d 86.8c 85.6cd 84.4bc 83.4b 83a 77.6a 73.1bc
WSAPC 93.1a 92a 91a 89.5a 86.4a 84.3a 79.5a 75.7a
WSPH-Thz 76.3g 74.5gh 71h 68.1i 66.9ef 64.4g 61.1f 51.7h
WSPH-Pflo 85.5e 84.5de 80.9e 78.1e 77.7c 74d 71.1d 67.2e
WSPHC 91.2b 89.9b 86.4c 83.9c 81.5b 78.1bc 74.3c 70.6d
Dry DSAP-Thz 74.2h 72.2i 71.5h 69.6hi 68e 65fg 62.6ef 58g
DSAP-Pflo 84.17f 83e 81.6e 80.3d 78.3c 77.3c 75.2bc 71.9cd
DSAPC 92.3ab 90.9ab 88.5b 86.2b 86.1a 82.3a 79.4a 74.4ab
DSPH-Thz 74.1h 74.2h 74.5g 71gh 68.1e 66.9f 64.4e 59.5fg
DSPH-Pflo 85.4e 84.1de 84.5d 80.9d 78.1c 77.7bc 74c 71.6cd
DSPHC 90.7c 89.5b 86.7c 84.8bc 81.2b 79.6b 77.2ab 75.8a
Control Thz 74.1h 70.1j 64.8i 59.5k 54.2h 48.9j 43.6i 38.3j
Pflo 85ef 78.2f 71.7h 65.3j 58.8g 52.4i 45.9h 39.5j
C 92.02b 85.6cd 79f 72.3fg 65.7f 59h 52.4g 45.7i
LSD 1.1 1.8 1.8 2.3 2.3 2.2 2.3 2.1
Means within a column followed by different letters are significantly different at 5% level of significance and those
following by the same letter do not significantly at 5% level of significance.
Table 4b: Bioefficacy of formulations with control at 25⁰ C storage temperature
% Inhibition
Type Formulation 0 day 15 day 30 day 60 day 90 day 120 day 150 day 180 day
Wet WSAP-Thz 76.2g 75.1fg 74.3e 71.2e 69.8e 66.1d 59.9cd 53.4cd
WSAP-Pflo 86.9d 86.4c 85.4b 82.1b 79.6b 76.0b 69.9b 65.3a
WSAPC 92.6a 92.1a 89.8a 87.2a 83.3a 80.5a 74.3a 66.0a
WSPH-Thz 75.9g 71.4h 66.6h 60.9g 55.7g 50.5g 44.5h 37.7 g
WSPH-Pflo 82.3f 79.7e 76.4e 70.7e 70.7de 62.4e 55.1e 47.0e
WSPHC 91.0bc 88.5b 83.4bc 78.4c 73.3cd 65.6d 57.1de 51.5d
Dry DSAP-Thz 73.4h 71.2h 68.9g 66.5f 60.1f 53.3f 47.6g 42.5f
DSAP-Pflo 82.9f 81.4de 79.7d 74.8d 70.1e 63.8de 58.5d 52.7cd
DSAPC 91.6abc 90.6a 85.6b 77.9c 74.9c 69.0c 62.4c 54.9bc
DSPH-Thz 73.5h 73.4g 71.4f 66.6f 60.9f 55.7f 50.5f 45.3e
DSPH-Pflo 84.7e 82.9d 79.7b 76.4cd 70.7de 70.7c 62.4c 56.2b
DSPHC 90.7c 86.2c 83.0bc 77.2cd 70.6e 66.2d 61.6c 56.7b
Control Thz 73.8h 66.7i 60.3i 53.8i 47.4i 40.9hi 34.5i 28.0h
Pflo 84.2e 76.8f 67.2gh 57.6h 48.0i 38.4i 28.8j 19.2j
C 91.8ab 80.8e 71.3f 61.8g 52.3h 42.8h 33.4i 23.9i
LSD 1.035 1.8008 2.31 2.71 2.61 2.69 2.90 2.56
Means within a column followed by different letters are significantly different at 5% level of significance and
those following by the same letter do not significantly at 5% level of significance.
82
Table 4c: Bioefficacy of formulations with control at 45⁰C storage
% Inhibition
Type Formulation 0 day 15 day 30 day 60
day
90 day 120
day
150
day
180
day Wet WSAP-Thz 75.6
g 73.1
g 71.7
e 67.3
f 63.2
d 57.5
d 49.6
c 36.2
e
WSAP-Pflo 85.6c
83.5c
81.8bc
76.5b
72.2b
65.4b
53.4b
41.5d
WSAPC 91.8a
90.8a
87.8a
82.6a
77.4a
72.1a
61.5a
53.6a
WSPH-Thz 75.0g
68.2i
62.2g
51.7h
43.3fg
36.1f
25.1h
17.7j
WSPH-Pflo 80.9f
78.1f
71.9e
62.7g
54.6e
46.1e
33.5f
22.8i
WSPHC 90.9ab
86.4b
80.3cd
72.7cd
68.6c
61.4c
51.4bc
45.1c
Dry DSAP-Thz 71.5i
69.8hi
60.7gh
54.2h
46.3f
36.7f
30.2g
24.5hi
DSAP-Pflo 80.7f
76.2f
71.1e
63.4g
54.0e
44.7e
38.2e
27.7g
DSAPC 91.2a
89.9a
83.3b
75.5bc
68.8c
61.5c
52.5bc
45.6bc
DSPH-Thz 72.9h
71.5gh
68.2f
62.2g
51.7e
43.3e
36.1ef
27.0gh
DSPH-Pflo 84.6d
80.7d
78.1d
71.9de
62.7d
54.6d
46.1d
31.4f
DSPHC 90.2b
83.3c
78.2d
69.2ef
60.5d
55.4d
52.9bc
48.4b
Control Thz 73.2h
64.2j
52.9i
41.7j
30.4i
19.2h
7.9j
2.4l
Pflo 83.2e
71.7gh
59.1h
46.6i
34.0h
21.5h
8.9j
4.3kl
C 91.0ab
78.4e
66.4f
54.3h
42.3g
30.2g
18.2i
6.1k
LSD 0.97 2.11 2.46 2.80 3.07 3.03 3.31 2.9
Means within a column followed by different letters are significantly different at 5% level of significance and
those following by the same letter do not significantly at 5% level of significance.
83
Figure 2: Relative reduction (%) in viability of bioagents in test formulations (A-C) vis-à-vis controls (D) at different storage temperatures
A
D C
B
83
GENERAL DISCUSSION
In Indian agriculture, horticultural crops play a major role having commercial and medicinal
uses. Unfortunately many crops suffer from many fungal and viral diseases. Amongst the
fungal diseases, the most serious one is color or root rot disease. It is caused by Pythium
aphanidermatum and Phytopthora parasitica etc. Problems from the use of chemical
fungicides are increasing due to the pollution in normal environment (Singh et al., 1995).
Biocontrol approach involving use of non-pathogenic antagonistic microorganisms especially
Trichoderma and Pseudomonas, and Bacillus, spp. is viewed as a potential ecofriendly
alternative. Some antagonistic microorganisms are commonly used. Naturally occurring
biocontrol results from combined action of antagonists rather than from a high population of
a single antagonist (Hornby, 1983; Lemanceau & Alabouvette, 1991; Chaube et al., 2003).
Compatibility evaluation in vitro of Trichoderma harzianum and Pseudomonas
fluorescens revealed 68% growth of T.harzianum and 88% growth of P.fluorescens in
presence of each other. Their combined bioefficacy (in vitro) against Pythium
aphanidermatum was 93% as compaered to individual bioefficacies (T harzianum 70% and
P. fluorescens 82%).
In order to harness the biocontrol potential of Trichoderma and Pseudomonas
species, various efforts have been made worldwide to formulate the bio-agent conidia and/
hyphae into inert carriers or liquid form. Temperature and moisture content play determinant
role in the shelf-life and viability of the bioformulations. To overcome these constraints, one
of the recent technologies include immobilization of wet or dry biomass within cross linked
polymers such as alginate and carrageenan as formulated pellets (Cho and Lee, 1999). In this
context, new generation carrier approach is use of a special class of polymers called
hydrogels. Hydrogels are of two types based on porosity and swelling rates, nonporous
superabsorbent polymers (SAP) and superporous hydrogels (SPH).
Guar gum based superabsorbent and super porous hydrogels were developed in the
present work and extensively studied in relation to their swelling and matrix properties.
The guar gum based superabsorbent hydrogels (GG-SAP) were prepared by
standardising various reaction parameters like monomer: backbone concentration, cross
84
linker concentration, initiator concentration, water volume per gram feed and alkali molar
ratios. The optimised hydrogel based on water absorbency was studied under different
temperatures, time periods, pH and water quality and also in salt and fertiliser solutions. QH₂O
increased significantly with increase in temperature from 10⁰ to 55⁰ C. Superior swelling
characteristics of GG-SAP at pH 4, 7 and 9 and more particularly 9, was displayed which
points toward the lead for a potential soil conditioner for the acidic and alkaline soils. As
compared to maximum QH₂O values of the product obtained in distilled and deionized water,
the swelling in tap water (EC = 2.04 mhos/ cm, pH = 7.7), hard water of different simulated
ionic strengths decreased, although to a small extent. The GG-SAP attained equilibrium water
absorbency in 14 hr at 50⁰ C, whereas at 25⁰ C the same was attained in 18 hr. In all the salt
solutions, absorbency was less compared with that in distilled water. The hydrogel exhibited
minimum reduction in QH₂O in urea solutions at all test concentrations.
The effect of GG-SAP addition on water absorption and retention capacity of sandy
loam and soilless medium under different temperatures and pressures showed that at both the
experimental temperatures (25⁰ C and 45⁰ C), the hydrogel amended soil and soilless medium
absorbed more water than respective controls. Amendment with hydrogel @ 0.75% exhibited
significantly higher water absorbency (WAC) than @ 0.5% in both the plant growth media.
As compared to control, the water holding capacity (WHC) of the hydrogel (GG-SAP)
amended soil, at both rates 0.5% and 0.75% remained higher at field capacity and also at all
matric tension respectively, though the difference between 0.5% and 0.75% amended soils
became narrower at matric tensions above 2.53 pF. Similar behaviour was observed for the
soilless medium. The hydrogel amended soilless medium @ 0.5% and 0.75% showed higher
WHC values (65.2% and 85.29%) respectively as compared to control. Irrespective of the
type of absorbent material, the percent moisture absorbed by free SAP is more relative to
when it is present in a plant growth medium. Addition of GG-SAP to soil and soilless
medium increased water availability to plant as compared to respective controls. At 4.2 pF
(corresponding to permanent wilting point) hydrogel amended soil released 21 to 37% more
water than control. Similar behaviour was observed in case of soilless medium.
Characterization of GG-SAP was done by FT-IR, scanning electron microscopy, Solid state
C13
NMR and XRD which revealed that guar gum was grafted and cross linked to convert in
to polymer.
85
In case of guar gum based porous hydrogels (GG-SPHs), various reaction variants
such as method of preparation, foam stabiliser concentration, porogen concentration, cross
linker concentration, initiator concentration, volume of water per unit feed, monomer
concentration, foaming aid type and reaction duration which influence the swelling
characteristics and network properties of superabsorbent hydrogels, were standardised. In
most of available reports on SPH such extensive investigations are lacking. Swelling
behaviour of the prepared SPH in response to pH, time period, temperature, salts and
fertilizers and water quality showed that at pH 9, the hydrogel showed maximum swelling.
As compared to deionized water, the absorbency was significantly reduced in different
aqueous environments. GG-SPH swelling was fast (50% absorbency attained in 30 minutes).
After 30 minutes, swelling ratio consistently increased and attained its maximum equilibrium
swelling in 6 hr. The porogen free SAP, on the other hand reached its QH₂O maximum in 24
hr. In urea solution, the absorbency exhibited fall with increase in its concentration from
5mM to 20mM, though the overall reduction in QH₂O was much lesser than that in other salt/
fertilizer solutions.
Change in density and porosity as a function of cross linker content exhibited ironical
trend which could be attributed to cross-linker concentration range chosen in the present
experiment. For the formation of network of optimum crosslinking density, a threshold
quantity of cross linker is necessary which is specific to the experimental conditions, back
bone nature and content etc. The minimum cross linker concentration screened in the present
study was in the range of 0.005-0.02 wt% and the maximum test concentrations was 0.7-0.8
wt%. The screened concentration range was much lower than that reported (Chavda et al.,
2010; Kabiri et al., 2003). SEM analysis of these developed hydrogels revealed porous
structure.
The carriers so prepared i.e. GG-SAP and GG-SPH were used for entrapment of
bioagents, Trichoderma harzianum and Pseudomonas fluorescens individually and as mixture
(1:1). The formulations were prepared in two ways i.e. dry method and wed method based on
their physical appearance. Hydrogel carriers of GG-SAP and GG-SPH were used to prepare
12 compositions containing T. harzianum and P. fluorescens alone and in combination.
Viability and antagonistic activity of formulated bioagents were tested periodically through
six months of storage period along with control.
86
Trichoderma harzianum and Pseudomonas fluorescens formulated singly or in
combinations in dry and wet formulations sustained significantly high viability (>108 c.f.us/g)
at all three storage temperatures. This noteworthy observation particularly at 450 C is hitherto
unknown in literature. Moisture availability around the microbial units could be one major
attribute (Beatrice et al., 1991, Cigdem and Merin, 2005, Walker et al., 2004). Nakeeran,
(2005) observed that higher water holding capacity of carrier is an essential characteristic of
good carrier.
All the combination formulations, irrespective of the type of carrier and method of
preparation exhibited >50% Pythium inhibition at all the three storage periods. At 50 C while
control treatments comprising T. harzianum and P.fluorescens alone and their mixture stored
for a period of 6 months showed 28-45% inhibition of Pythium, the test formulations
exhibited percentage inhibition in the range of 52% to 76%. Similarly at 250 C, all the test
formulations exhibited significantly higher bioefficacy. In particular, WSAP formulations of
individual bioagents as well as their mixture performed best with 53-66% reduction in the
pathogen growth. Bioefficacy of formulations exhibited gradual decline after 4 months of
storage at 450C. The percentage inhibition caused by combination formulations WSAP-C,
DSAPC, WSPH-C and DSPH-C at all temperatures across 180 days during storage period,
was significantly higher as compared to the corresponding formulation of individual T.
harzianum and P. fluorescens (WSAP-Thz, WSAP-Pflo, DSAP-Thz, DSAP-Pflo, WSPH-Thz,
WSPH-Pflo, DSPH-Thz, DSPH-Pflo). A gradual reduction in the observed bioefficacy of all
the test formulations was observed at higher storage temperature. At 450C, drastic reduction
in the percentage inhibition (<50%) was observed in all the formulations except WSAP-C
(53.6%). However, at 5 month storage period all the combinations exhibited 51.4 % - 61.5 %
inhibition of Pythium.
Based on the viability data of the test formulations on 180th
day at all the three storage
temperatures, the bioefficacy was expected to be high (>50%). Lower bioefficacy values
indicate towards need to standardize the method of application and the quantity of
bioformulation to be applied in each replicate.
87
SUMMARY AND CONCLUSION
1. Agriculture continues to play a major role in Indian economy; contributing
approximately 12.1% of the total national gross domestic production (GDP).
Horticultural crops are front runners for betterment of small and marginal farmers.
2. Many horticultural crops suffer from many fungal and viral diseases. Amongst the
fungal diseases, the most serious one is color or root rot disease caused by Pythium
aphanidermatum.
3. Pythium is being controlled by mainly two approaches- chemical and biological.
Biocontrol approach with the help of antagonistic microorganisms, especially
Trichoderma and Pseudomonas, and Bacillus, spp., offers an ecofriendly alternative
to chemical control.
4. Naturally occurring biocontrol results from combined action of antagonists rather than
from a high population of single antagonist. Strategy of combining two or more
antagonists to enhance the level of disease management is imperative.
5. Serious bottlenecks in full realization of biocontrol potential are high temperature,
limited moisture in the soil and requirements high inoculation rates of the desired
organism. bioformulations containing bioagents entrapped in the carriers hold promise
in this context.
6. Recent technologies for the formulation of biocontrol organisms involve
immobilization of wet or dry biomass within cross linked polymers such as alginate
and carrageenan.
7. Hydrogels, the cross linked speciality polymers with versatile network and water
absorption characteristics have been explored worldwide to serve as carriers of
agrochemicals.
8. The objective of present work was to entrap Trichoderma and Pseudomonas
(effective against Pythium) individually or in combinations in the matrix of hydrogels.
9. Novel biopolymeric hydrogels GG-SAP and GG-SPH (superabsorbent and
superporous hydrogels respectively) based on guar gum were prepared to explore
their potential as carriers of microbes.
88
10. Swelling response of GG-SAP and GG-SPH was studied in different simulated
environments like temperature, time period, pH, salt type and fertilizers at different
strengths.
11. The hydrogels based on water absorbency were characterised by FT-IR, solid state
C13
NMR, XRD and scanning electron microscopy and elemental analysis.
12. Water absorption and retention capacity of GG-SAP on addition to plant growth
media was studied.
13. Density and porosity variation of GG-SPHs with cross linker concentration was
studied. Increase in cross linker concentration resulted in decrease in porosity and
increase in density, a hitherto unknown observation not reported in literature.
14. The SAP and SPH with optimized properties were used as carrier for bioagents.
15. Thz and Pflo exhibited 68% and 88% growths respectively in presence of each other
(in vitro).
16. Two types of formulations dry and wet were prepared employing T. harzianum and P.
florescence, each alone and as mixture (1:1). A total of twelve compositions were
prepared and preserved at three temperatures (5⁰, 25⁰ and 45⁰ C). The details of the
preparation of formulations will be protected under IPR.
17. The prepared compositions were evaluated for shelf life characteristics in terms of
viability as a function of storage temperature.
18. All compositions maintained >5×108
CFUs of each bioagent per gram carrier up to
180 days.
19. In vitro antagonistic evaluation of prepared formulations against Pythium
aphanidermatum showed >50% inhibition exhibited by combined formulations at all
temperatures up to 180 days and individual formulations showed up to 50%
inhibition for 150 days.
20. It is concluded that hydrogel based microbial formulations can be successfully
developed for management of Pythium. The findings of the present study however
need to be standardised in terms of method of application and dose of application of
prepared formulations under field conditions.
i
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FORM 2
THE PATENT ACT, 1970
(39 OF 1970)
THE PATENTS RULES, 2003
TENTATIVE TITLE OF THE INVENTION:Novel biopolymeric hydrogel carriers and the
process of making novel bioformulation based there upon with enhanced shelflife characteristics
ABSTRACT
Novel, ecofriendly indigenous, easy to use mixed bioformulations with enhanced shelf-life at
otherwise unfovourable high temperatures characteristic of tropics, characterized by one or more
strains, species or genera of biocontrol agents immobilized and entrapped inside the matrix of
novel water insoluble or soluble nonporous and/or porous hydrogels of semi-synthetic origin
maintained at moisture content equivalent of an appropriate per cent of the water absorption
capacity of the superabsorber, capable to withstand temperature in the range 0-500C for an
effective time period of 36 to 9 months respectively with 50-100% survival of the entrapped
microbes even in the absence of any extraneous source of nutrients, providing favourable
environment for microbes to retain their viability as such, free from microbial attack induced
degradation has been developed by simple yet novel method/sof entrapment of microbes into the
carrier matrix under ambient conditions, with additional advantages of transparent medium for
direct examination of microbial cells under microscope and retaining water in the medium to
which it is applied. The formulation can be easily applied in the soil, soilless media, seed and the
like without requiring any chemical reagent.
Synthesis and evaluation of guar gum based hydrogels as carriers of bio agents
for Pythium management
ABSTRACT
Fungal diseases pose a major challenge in the productivity of fruits and vegetables. Root
rot disease, caused by Pythium aphanidermatum is of major concern. In view of the
environmental concerns related to crop protection chemicals, biocontrol approaches for pest
management are of current interest in crop protection programmes. However, the potential of
bioagents remains underutilized due to shelf and field life constraints such as high
temperature and limited moisture in the soil. Development of suitable formulations to
overcome these constraints is thus, imperative. The main objective of present thesis was to
develop biopolymeric hydrogels based bioformulations of Trichoderma harzianum and
Pseudomonas fluorescens and to evaluate their shelf lives and bioefficacy against Pythium
aphanidermatum under laboratory conditions.
Guar gum (GG), a galactomannan polysaccharide of plant origin was employed to
prepare cl-GG-g-polyacrylate superabsorbent (GG-SAP) and novel superporous hydrogels
(GG-SPH) as carrier materials. Reaction parameters such as backbone particle size,
backbone-monomer ratio, cross linker and initiator concentrations, volume of water per unit
feed, foaming aid, porogen, foam stabilizer etc. were optimized. Microwave synthesis
compared with the thermally initiated polymerization technique was found uneconomical
under the process conditions employed. Structures of hydrogels were established by FT-IR
and solid state 13
C NMR spectroscopy, X-ray diffraction (XRD) and scanning electron
microscopy (SEM). Swelling behaviour of the representative GG-SAP and GG-SPH was
evaluated w.r.t. ionic strengths of salt and fertilizer solutions, temperature, water quality and
pH under laboratory conditions. GG-SAP exhibited significantly high swelling in acidic and
alkaline environments as well as in the presence of salts and fertilizers. GG-SPH exhibited its
pH-sensitive behaviour and faster swelling rate as compared to the corresponding GG-SAP.
Addition of GG-SAP to plant growth media (sandy loam soil and soilless medium)
significantly improved their moisture characteristics.
GG-SAP and GG-SPH with optimized matrix properties were used to prepare 12
novel bioformulations of Trichoderma harzianum and Pseudomonas fluorescens. All the test
compositions contained > 5×108 colony forming units per gram of carrier at three different
storage temperatures, 5⁰, 25⁰ and 45⁰ C for a study period of 180 days. Bioefficacy
evaluation (in vitro) of test compositions stored at different temperatures, against Pythium
aphanidermatum showed that the test compositions stored at 5⁰ and 25⁰ C were able to
inhibit >50% Pythium population (in vitro) up to 180 days storage period. Compositions
stored at 45⁰ C exhibited >50% (or equal) inhibition up to 90 days storage period. Finding of
the present work point towards a promising hydrogel based bioformulation approach for
integrated water and disease management in horticultural crops, which will be further,
established under practical use conditions.
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