Monocrotophos. Dicofol. Dimethyl (E)-1-methyl-2-(methylcarbamoyl)vinyl phosphate. 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol. Figure â 4 Structure and ...
Kinetic biosorption studies of chemical pesticides by microbial biomass isolated from agricultural soils
Dissertation submitted in partial fulfilment for the degree of Master of Science in Biotechnology Submitted By Aradhana Basu Roll No: 1261006
KIIT School of Biotechnology, Campus- 11 KIIT University Bhubaneswar, Odisha, India
Under the Supervision of Mr. Ritesh Pattnaik Assistant Professor School of Biotechnology KIIT University
Dr. Suraj Kumar Tripathy Assistant Professor School of Biotechnology KIIT University
KIIT School of Biotechnology, Campus- 11 KIIT University Bhubaneswar, Odisha, India
CERTIFICATE This is to certify the dissertation entitled “ Kinetic biosorption studies of chemical pesticides by microbial biomass isolated from agricultural soils” submitted by Aradhana Basu in partial fulfillment of the requirement for the degree of Master of Science in Biotechnology, School of Biotechnology, KIIT University, Bhubaneswar bearing Roll No. 1261006 & Registration No. 12363725778 is a bonafide research work carried out by her under my guidance and supervision from January 2014 to May 2014.
Supervisor
(Mr. Ritesh Pattnaik)
Co-supervisor
(Dr. Suraj Kumar Tripathy)
CERTIFICATE This is to certify that the dissertation entitled “ Kinetic biosorption of chemical pesticides by microbial biomass isolated from agricultural soils “ submitted by Aradhana Basu, Roll No. 1261006, Registration No. 12363725778 to the KIIT School of Biotechnology, KIIT University, Bhubaneswar-751024, for the degree of Master of Science in Biotechnology is her original work, based on the results of the experiments and investigations carried out independently by her during the period from Jan’14 to May’14 of study under my guidance. This is also to certify that the above said work has not previously submitted for the award of any degree, diploma, fellowship in any Indian or foreign University.
Supervisor
(Mr. Ritesh Pattnaik)
Co-supervisor
(Dr. Suraj Kumar Tripathy)
ACKNOWLEDGEMENT I express my sincere obligations to Dr. Mrutyunjay Suar, Director, School of Biotechnology, KIIT University for permitting me to conduct my research work in this highly esteemed institute. I express my sincere and deepest sense of gratitude to my advisor, supervisor and guide Mr. Ritesh Pattnaik, Assistant Professor, School of Biotechnology, KIIT University and Dr. Suraj Kumar Tripathy, Assistant Professor, School of Biotechnology, KIIT University for their constant supervision, scholarly guidance, constructive criticism, advice and help throughout the period of my work and preparing this thesis. Their kind attention has made my research a memorable experience. I thank with profound honour and regards to Dr.Amrita Mishra for her valuable suggestions and help. I express my gratitude and thanks to Mr. Sourav Das for his suggestions, help and great co-operation during my work. I am also thankful to Mr. Sayantan Sinha and Mr. Bhaskar Das for their help and co-operation. Lastly I thank almighty and my parents for their encouragement, moral support and blessings showered upon me without which the present investigation would not have been successful.
Date:
Aradhana Basu
CONTENTS
1. ABSTRACT
1
2. INTRODUCTION
2-4
3. REVIEW OF LITERATURE
5-25
4. MATERIALS AND METHOD
26-32
5. RESULTS AND DISCUSSION
33-45
6. CONCLUSION
45
7. REFERENCES
46
LIST OF FIGURES AND TABLES
Figure 1
Representation of a typical pesticide cycle in an ecosystem
Figure 2
Classification of common pesticides
Figure 3
Representation of consumption chart of pesticides
Figure 4
Structures and IUPAC name of pesticides used in the study
Figure 5
Fate of pesticides in environment
Figure 6
Langmuir isotherm
Figure 7
Cultivation and isolation of microbes using microbial techniques from soil samples
Figure 8
Biochemical tests for isolated microorganisms
Figure 9
Reduction potential assay of pesticides by disc diffusion assay
Figure 10
Adsorption of pesticides with and without heat treatment
Figure 11
Kinetic biosorption studies of different pesticides
Figure 12
Kinetic biosorption studies of different pesticides at different pH
Figure 13
Kinetic biosorption studies of different pesticides at different agitation speed
Table -1
Classes of pesticides
Table -2
Data on biosorption of pesticides by various microorganisms
Table -3
Physico-chemical properties of different soils used in the study
Table -4
Morphological characteristics of isolated microbial samples
ABBREVIATIONS OD
Optical density
0
Degree Celsius
g
gram
mg
Milligram
l
Litre
µl
Microlitre
mM
Millimolar
min
Minute
rpm
Revolutions per minute
nm
Nanometer
NA
Nutrient agar
NB
Nutrient Broth
CZA
Czapek dox agar
Strep
Streptomycin
C
ABSTRACT
The present study primarily focuses on screening of indigenous microbes from different agricultural tracts of Odisha having effective remedial potential. For better productivity, agricultural soils are being still supplemented with toxic chemical pesticides on regular basis. Isolation of indigenous microbes having pesticide degradation and or adsorption potential are done using microbiological enrichment methodologies followed by characterization to establish their biosorption ability. Investigations regarding the adsorption efficiency of microbes and the influence of different factors such as pH, rpm were studied. Results establish that increase in pH and agitation speed enhances the adsorption efficiency by about 80 % under without heat treatment conditions of adsorbate as compared to heat treated. The use of mixed microbial consortia including bacterial and fungal biomass plays a critical role for adsorbing toxic chemicals including pesticides present in varying concentrations in soil and sediments.
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Chapter 1 Introduction In the early part of 19th century, there was a rapid expansion of urbanization including various chemical manufacturing industries which led to environmental pollution events. Several dramatic accidents of chemical contamination and toxicity in 1970 and 2010 made the perception of environmental deterioration flourish. Environmental risks such as diffuse pollution, particularly long-range transport of atmospheric pollutants, exemplified by acid deposition, was identified as serious environmental and ecological issues. The release of harmful chemicals including methyl isocyanides (MIC) into the environment can be considered as one of the worst examples of disaster and widespread pollution. During the past twenty years, concern has arisen due to the presence of pesticides in the environment and the threat they pose to wildlife and mankind. Chemical pesticides have contributed greatly to the increased yields in agriculture by controlling pests and diseases and also towards checking the insect-borne diseases (malaria, dengue, encephalitis, filariasis, etc.) in the human health sector. The need to increase world food production for the rapidly growing population is well recognized. However, the sporadic use often leads to significant consequences not only in public health but also in food quality, resulting in an impact load on the environment and hence the development of pest resistance. Inappropriate application of pesticides affects the whole ecosystem by entering the residues in the food chain and polluting the soil, air, ground, and surface water. The term “pesticide” is used to define a variety of agents that are classified on the basis of their potential to kill living organisms. They include insecticides, herbicides, and fungicides. As they are effective in controlling living species, pesticides are biologically active substances which are ubiquitously present in the living environment, and lead into various health risks and disorders. Pesticides are characterized by their unique chemical structure, patterns of use by the society and their interaction with the environment. Pesticide structures are developed to mimic and therefore substitute for specific molecules in targeted biological reactions. They can be classified mainly by considering two criteria: chemical classes and target organism. Humans are exposed to pesticides by different routes of exposure such as inhalation, ingestion, and dermal contact. 2 | P a g e
Increasingly, acute exposures to pesticides were illustrated as vulnerable to a wide spectrum of immunosuppression, neurobehavioral disorders, autoimmune disease, and reproductive abnormalities. Pesticide pollution to the local environment also affects the lives of birds, wildlife, domestic animals, fish, and livestock. The general population is exposed to the residues of pesticides, including physical and biological degradation products in air, water, and food. On the other hand, use of un-prescribed pesticides in inappropriate doses not only disturbs the soil conditions but also the healthy pool of bio-control agents that normally co-exist with the vegetation. Throughout history, various types of pests, such as insects, weeds, bacteria, rodents, and other biological organisms, have affected or threatened human health because of their use for thousands of years to control the pests. Today, more than 500 different formulations of pesticides are being used in the environment, and agriculture holds the largest single share of pesticides use. The removal of pesticides from soil and water is one of the major environmental concerns today. In past few years presence of pesticide residue in the agricultural soil and ground water resources has grown significantly and has become an intensive issue related to environment and ecosystem. The wide range of pesticides in use makes research extremely difficult for producing a single method for the removal of pesticides that applies universally. Several methods are available for pesticide removal including photocatalytic degradation, combined photo-Fenton and biological oxidation, advanced oxidation processes, aerobic degradation, nanofiltration membranes, ozonation, coagulation, fluid extraction, solid phase extraction, and adsorption. In recent times, adsorption of pesticides on carbonaceous, lignocellulosic and polymeric materials from aqueous solutions has been extensively studied. Adsorption is a well-known equilibrium separation process and an effective method for soil and water decontamination application. Adsorption has been found to be superior to other techniques for water re-use in terms of initial cost, flexibility, and simplicity of design, ease of operation, and insensitivity to toxic pollutants. Reports suggest that both living and dead (heat killed, dried, acid, and/or otherwise chemically treated) biomass can be used to remove pesticides, but maintaining a viable biomass during adsorption is difficult as it requires a continuous supply of nutrients and avoidance of organic toxicity to the microorganisms. The use of dead microbial cells in 3 | P a g e
biosorption is more advantageous for soil and water treatment because dead organisms are unaffected by toxic wastes and do not require a continuous supply of nutrients, and moreover they can be regenerated and reused for many cycles. Dead cells have been shown to accumulate pollutants to the same or greater extent of growing or resting cells. The mechanism of binding by inactivated biomass may depend on the chemical nature of pollutant (species, size, ionic charge), type of biomass, its preparation and its specific surface properties, and environmental conditions (pH, temperature, ionic strength, existence of competing organic or inorganic ligands in solution) A wide variety of microorganisms including aerobic microbes and fungal species are known to utilize organic pesticides as the sole carbon or energy source, such as Pseudomonas pickettii, Alcalilgenes eutrophus, Desulfomonile tiedjei, Phanerochaete chrysosporium. Investigations on adsorption behavior of different classes of pesticides including the organophosphorous pesticides such as lindane and malthion on Rhizopus oryzae biomass indicate that the adsorption process is independent on the pH of the solution or incubation temperature. Hydrophobic interaction is mainly responsible for this adsorption process. It can be used for the adsorptive removal of pesticides from wastewater containing different matrices. Moreover, adsorption process is influenced by a number of factors, such as adsorbent dose and size, contact time, agitation speed, temperature, pH, and ionic strength of the aqueous solution. The use of microbial biomass would be effective enough in controlling the toxicity of pesticides present in environment. Considering the above information available on biosorption of pesticides in soil and water, the present work is envisaged with the following objectives: 1. Isolation of indigenous microbes from agricultural soil samples having pesticide sorption potential. 2. To study the kinetics of biosorption of selected pesticides using isolated microbial biomass. 3. To examine the effect of factors such as pH, agitation time and adsorbent dose on biosorption of pesticides.
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Chapter – 2 Review of Literature The use of pesticides is ubiquitous in modern agriculture and is important to increase crop yield and reduce post-harvest losses. However, indiscriminate and excessive use of agricultural pesticides can lead to contamination of land and water causing serious problems. The extensive and massive use of chemical pesticides in agriculture activities has serious impacts on the environment, compromising soil and water quality. As several pesticides are used simultaneously on most agricultural crops, it often leads to a higher risk and increased pollution. Emissions of pesticides in the environment are generally divided into diffuse and direct losses. Diffuse contamination via percolation, runoff, drainage and drift, explains only a part of the pesticides that reach surface and groundwater. Point-source contamination by pesticides has been identified as a major concern contributing significantly to the deterioration of natural water resource quality. Indeed, several studies demonstrated that 40–90% of surface water contamination is attributable to point-source contamination produced by improper pesticide handling before or after their field application. Although pesticides are intensively used in agriculture to enhance productivity, yet much effort is required to manage and reduce possible deleterious effects on the environment. The soil compartment has a major influence on the fate and behavior of pesticides applied to crops preemergence or early postemergence or chemicals subject to wash off from crop surfaces. Once in the soil, pesticide molecules partition between the aqueous and solid phases, which affects many other aspects of their behavior: sorption can be rate limiting to volatilization, bioavailability (and thus efficacy and biodegradation rate), and subsurface transport. Understanding the fate of a pesticide in soil is fundamental to the accurate assessment of its environmental behavior and vital in ensuring the safe use of new and existing products. It is also necessary to develop and validate computer simulation models for use as predictive tools in future environmental fate assessments. Developing countries including India often rely on farming and agriculture. For thousands of years, agricultural was a natural process that did not harm the land it was done on. In fact, 5 | P a g e
farmers were able to pass down their land for many generations and it would still be fertile as ever. The economy of India is largely dependent on the quality and quantity of its agricultural production. However, modern agricultural practices have started the process of agricultural pollution. This process causes the degradation of the eco-system, land and environment due to the modern day by-products of agriculture. Harvests require intensive cultivation, irrigation, fertilizers, and what is more important the use of chemicals to protect plants from pests and plant diseases. Pesticides often known as protection products, are used to protect plants from damaging influences such as weeds, plant diseases or insects. This use of pesticides is so common that the term pesticide is often treated as synonymous with plant protection product, although it is in fact a broader term, as pesticides are also used for non-agricultural purposes. The term pesticide includes all of the following: herbicide, insecticide, insect growth regulator,nematicide, termiticide, molluscicide, piscicide, avicide, rodenticide, predacide, bactericide,
insect
repellent,
animal
repellent,
antimicrobial,
fungicide,
disinfectant
(antimicrobial), and sanitizers Pesticides applied to soil are subject to a complex web of many interacting physical, chemical and biological processes. Models are therefore potentially powerful tools to test and improve understanding of the environmental fate of pesticides (Boesten, 2000). Degradation is one of the most sensitive model parameters determining the fate of pesticides in soil and losses to surface water and groundwater (Boesten, 1991; Dubus et al., 2002). Variation of pesticide degradation is therefore especially critical in the context of risk assessment using fate models since it introduces considerable uncertainty into model predictions (Dubus et al., 2003; Lindahl et al., 2008).
2.1 Pesticides and their different classes Pesticides have numerous beneficial effects including crop protection, preservation of food and materials and prevention of vector-borne diseases. Pesticides include herbicides for destroying weeds and other unwanted vegetation, insecticides for controlling a wide variety of insects, fungicides used to prevent the growth of molds and mildew, disinfectants for preventing the spread of bacteria, and compounds used to control mice and rats.
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There is a large variety of pesticides designed to kill specific pests – those most widely used are listed below. Insecticides
(for killing insects) such as organochlorines, organophosphates and carbamates
Herbicides or
(e.g. paraquat, glyphosate and propanil).
weedkillers Fungicides
(to kill mould or fungi): when applied to wood, they are called wood preservatives.
Rodenticides
(to kill mice, rats, moles and other rodents).
Algicides
Control algae in lakes, canals, swimming pools, water tanks, and other sites.
Antifouling agents
Kill or repel organisms that attach to underwater surfaces.
Antimicrobials
Kill microorganisms (such as bacteria and viruses).
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Attractants
Attract pests (for example, to lure an insect or rodent to a trap). (However, food is not considered a pesticide when used as an attractant.)
Biopesticides
Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals.
Biocides
Kill microorganisms
Fumigants
Produce gas or vapor intended to destroy pests in buildings or soil.
Miticides
Kill mites that feed on plants and animals.
Microbial
Microorganisms that kill, inhibit, or out compete pests, including insects
pesticides
or other microorganisms.
Molluscicides
Kill snails and slugs
Nematicides
Kill nematodes (microscopic, worm-like organisms that feed on plant roots).
Ovicides
Kill eggs of insects and mites
Pheromones
Biochemicals used to disrupt the mating behavior of insects.
Repellents
Repel pests, including insects (such as mosquitoes) and birds.
Rodenticides
Control mice and other rodents.
2.11 Organophosphate pesticides Organophosphates affect the nervous system by disrupting the enzyme that regulates acetylcholine, a neurotransmitter. Most organophosphates are insecticides. They were developed during the early 19th century, but their effects on insects, which are similar to their effects on 8 | P a g e
humans, were discovered in 1932. Some are very poisonous (they were used in World War II as nerve agents). However, they usually are not persistent in the environment. 2.12 Carbamate pesticides Carbamate pesticides affect the nervous system by disrupting an enzyme that regulates acetylcholine, a neurotransmitter. The enzyme effects are usually reversible. There are several subgroups within the carbamates.
2.13 Organochlorine insecticides They were commonly used in the past, but many have been removed from the market due to their health and environmental effects and their persistence (e.g., DDT and chlordane).
2.14 Pyrethroid pesticides They were developed as a synthetic version of the naturally occurring pesticide pyrethrin, which is found in chrysanthemums. They have been modified to increase their stability in the environment. Some synthetic pyrethroids are toxic to the nervous system. 2.15 Sulfonylurea herbicides Includes nicosulfuron, triflusulfuron methyl and chlorsulfuron broad-spectrum herbicides that kill plants by inhibiting the enzyme acetolactate synthase. In the 1960s, more than 1 kg/ha (0.89 lb/acre) crop protection chemical was typically applied, while sulfonylureates allow as little as 1% as much material to achieve the same effect.
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Figure – 1 Representation of a typical pesticide cycle in an ecosystem. Pesticides are often referred to according to the type of pest they control. Pesticides can also be considered as either biodegradable pesticides, which will be broken down by microbes and other living beings into harmless compounds, or persistent pesticides, which may take months or years before they are broken down: it was the persistence of DDT, for example, which led to its accumulation in the food chain and its killing of birds of prey at the top of the food chain. Another way to think about pesticides is to consider those that are chemical pesticides or are derived from a common source or production method.
2.2 Commonly used pesticides in agriculture Chlorpyrifos [O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl)- phosphorothioate] is one of the most widely used organophosphate pesticides. Itwas first developed by the Germans in the 1930s and first introduced in 1965 in the USA as a home and garden insecticide by Dow Chemicals (Worthing, 1979). Chlorpyrifos is a non-systemic insecticide, which is effective against a wide range of insect pests of economically important crops (Fang et al., 2006). It enters into an insect body by contact and ingestion, and is also absorbed through the gut, skin and pulmonary membranes (Simon et al., 1998). Usually, it affects the nervous system of the target insects by inhibiting the activity of acetylcholinesterase by phosphorylation, both at the synapse of neurons 10 | P a g e
and in the plasma (Hui et al., 2010). As a result, acetylcholine is accumulated at the neuron synapse which causes the death of the target insect. It has been documented that organophosphate pesticides account for about 38% of the total pesticides used worldwide (Singh and Walker, 2006). A considerable amount of the pesticide either accumulates in the soil or enters intowater bodies after application. Unfortunately, less than 0.1% of the total applied pesticide reaches the target and the rest remains in the environment (Pimentel, 1995). Chlorpyrifos residues were detected up to eight years after application for termite treatment in 16 houses in North Carolina (Wright et al., 1994). Living organisms are exposed to pesticide residues in soil and water, resulting in a risk to the ecological balance (Kulshrestha and Kumari, 2011). There are also some reports on chlorpyrifos residues in the food chain (Aysal et al., 2004; Chandra et al., 2010). Several ecosystems across the world have been reported to be contaminated as a result of indiscriminate use of organophosphate pesticides, causing poisoning of millions of people and over 200,000 deaths annually.
Figure – 2 Classification of common pesticides
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Malathion [s-(1,2 -dicarbethoxyethyl)-o,o-dimethldithiophosphate), is an organophosphate pesticide widely used in public recreation areas, residential landscaping, and agricultural settings. Malathion itself is of low toxicity, it mainly concentrates in peel and may not readily removed by washing in water alone but easily enter the body through ingestion, inhalation and absorption through the skin results in its metabolism to malaoxon which is substantially more toxic. Longterm exposure to oral ingestion of malaoxon in rats, showed 61 times more toxic than malathion. It is cleared from the body quickly, in three to five days. Malathion degradation products include dimethyl phosphate, dimethyldithiophosphate, dimethylthiophosphate, isomalathion, malaoxon and due to cutinase, carboxylesterase, phosphatase enzymatic activity, malathion is degraded into malathion mono and dicarboxylic acid. Malathion kills insects and other animals, including humans, by inhibiting the acetylcholinesterases (AChE) that breaks down acetylcholine a chemical essential in transmitting nerve impulses across junctions between nerves, thus prolonging action potential in nerves, causing spasma, incoordination, convulsions, paralysis and ultimately death. The World Health Organization estimates that 500,000 pesticides poisoning cases occur annually in the world and that 1% are fatal (5000 death/year) prolonging action potential in nerves, causing spasma, incoordination, convulsions, paralysis and ultimately death. The World Health Organization estimates that 500,000 pesticides poisoning cases occur annually in the world and that 1% are fatal (5000 death/year)
2.3 Role of Pesticides in India In India, the production and use of pesticides started in 1952 and currently is the second largest manufacturer of pesticides in Asia after China and ranks twelfth globally (Mathur, 1999). There has been a steady growth in the production of technical grade pesticides in India which is about 102,240 metric tons comprising about 2% of the total world market. The pattern of pesticide usage in India is different from that for the world in general. The figure indicates the scenario where 76% of the pesticide used represents insecticide, as against 44% globally (Mathur, 1999). The use of herbicides and fungicides is correspondingly less heavy. The main use of pesticides in India is for cotton crops (45%), followed by paddy and wheat.
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Figure -3 Representation of consumption chart of pesticides
Malathion Chlorpyrifos
Diethyl 2‐[(dimethoxyphosphorothioyl)sulfanyl]butanedioate O,O‐Diethyl O‐3,5,6‐trichloropyridin‐2‐yl phosphorothioate
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Monocrotophos Dicofol Dimethyl (E)‐1‐methyl‐2‐(methylcarbamoyl)vinyl phosphate 2,2,2‐trichloro‐1,1‐bis(4‐chlorophenyl)ethanol Figure – 4 Structure and IUPAC name of pesticides used in the study
2.4 Harmful effects of pesticide toxicity Pesticides have been linked to a wide range of human health hazards, ranging from short-term impacts such as headaches and nausea to chronic impacts like cancer, reproductive harm, and endocrine disruption. Acute dangers - such as nerve, skin, and eye irritation and damage, headaches, dizziness, nausea, fatigue and systemic poisoning - can sometimes be dramatic, and even occasionally fatal. Chronic health effects may occur years after even minimal exposure to pesticides in the environment, or result from the pesticide residues which we ingest through our food and water. In July 2007 a study was conducted by researchers at the Public Health Institute, the California Department of Health Services, and the UC Berkeley School of Public Health to establish a result of six fold increase in risk factor for autism spectrum disorders (ASD) for children of women who were exposed to organ chlorine pesticides. Pesticides can cause many types of cancer in humans. Some of the most prevalent forms include leukemia, non-Hodgkins lymphoma, brain, bone, breast, ovarian, prostate, testicular and liver cancers. Studies by the National Cancer Institute found that American farmers, who in most respects are healthier than the population at large, had startling incidences of leukemia, Hodgkins disease, non-Hodgkins lymphoma, and many other forms of cancer. There is also mounting evidence that exposure to pesticides disrupts the endocrine system, wreaking havoc with the complex regulation of hormones, the reproductive system, and embryonic development. Endocrine disruption can produce infertility and a variety of birth defects and developmental defects in offspring, including hormonal imbalance and incomplete sexual development, impaired brain development, behavioural disorders, and many others. 14 | P a g e
Examples of known endocrine disrupting chemicals which are present in large quantities in our environment include DDT (which still persists in abundance more than 20 years after being banned in the U.S.), lindane, atrazine, carbaryl, parathion, and many others. 2.5 Fate of pesticides in the environment The extent of adsorption depends on the The fate of pesticides is affected not only by its own physicochemical properties but also by characteristics of the soil, management practices and environmental conditions (Halimah et al ,2010). Pesticides are distributed in the solid, liquid and gaseous phases in the vadose zone after their application depending upon the constant of adsorption, desorption and volatilization (Marino et al., 2002). The applied pesticides binds to plants, soil particles or sediments (Gebremariamet al., 2012). After a certain period of time its major fraction is either volatilized, hydrolyzed or biodegraded. Volatilization from soil depends on a number of factors such as concentration. Adsorption is probably the most important mode of interaction between soil and pesticides and controls the concentration of the latter in the soil liquid phase. Adsorption processes may vary from complete reversi-bility to total irreversibility properties of soil and the compound, which include size, shape, configuration, molecular structure, chemical functions, solubility, polarity, and charge distribution of interacting species, and the acid- base nature of the pesticide molecule (Bailey and White,1970; Senesi, 1992; Pignatello and Xing, 1996).Adsorption may be purely physical, as with van der Waals forces, or chemical in nature, as with electrostatic interactions. Chemical reactions between unaltered pesticides or their metabolites often lead to the formation of stable chemical linkages, resulting in an increase in the persistence of the residue in soil, while causing it to lose its chemical identity.
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Figure – 5 Fate of pesticides in the environment 2.6 Controlling the harmful effects of pesticide use –physical, chemical and biological methods In recent times, adsorption of pesticides on carbonaceous, lignocellulosic and polymeric materials from aqueous solutions has been extensively studied. Adsorption is a well-known equilibrium separation process and an effective method for soil and water decontamination application. Adsorption has been found to be superior to other techniques for water re-use in terms of initial cost, flexibility, and simplicity of design, ease of operation, and insensitivity to toxic pollutants. Reports suggest that both living and dead (heat killed, dried, acid, and/or otherwise chemically treated) biomass can be used to remove pesticides, but maintaining a viable biomass during adsorption is difficult as it requires a continuous supply of nutrients and avoidance of organic toxicity to the microorganisms. The use of dead microbial cells in biosorption is more advantageous for soil and water treatment because dead organisms are unaffected by toxic wastes and do not require a continuous supply of nutrients, and moreover they can be regenerated and reused for many cycles. Dead cells have been shown to accumulate pollutants to the same or greater extent of growing or resting cells. The mechanism of binding by inactivated biomass may depend on the chemical nature of pollutant (species, size, ionic charge), type of biomass, its preparation and its specific surface properties, and environmental conditions (pH, temperature, ionic strength, existence of competing organic or inorganic ligands in solution) 16 | P a g e
The wide range of pesticides in use makes research extremely difficult for producing a single method for the removal of pesticides that applies universally. Several methods are available for pesticide removal including photocatalytic degradation, combined photo-Fenton and biological oxidation, advanced oxidation processes, aerobic degradation, nanofiltration membranes, ozonation, coagulation, fluid extraction, solid phase extraction, and adsorption 2.7 Adsorption Adsorption is a process that occurs when a gas or liquid solute accumulates on the surface of a solid or a liquid (adsorbent), forming a molecular or atomic film (the adsorbate). It is different from absorption, in which a substance diffuses into a liquid or solid to form a solution. The term sorption encompasses both processes, while desorption is the reverse process. Adsorption is operative in most natural physical, biological, and chemical systems, and is widely used in industrial applications such as activated charcoal, synthetic resins and water purification. Similar to surface tension, adsorption is a consequence of surface energy. In a bulk material, all the bonding requirements (be they ionic, covalent or metallic) of the constituent atoms of the material are filled. But atoms on the (clean) surface experience a bond deficiency, because they are not wholly surrounded by other atoms. Thus it is energetically favourable for them to bond with whatever happens to be available. The exact nature of the bonding depends on the details of the species involved, but the adsorbed material is generally classified as exhibiting physisorption or chemisorption. Physisorption or physical adsorption is a type of adsorption in which the adsorbate adheres to the surface only through Van der Waals (weak intermolecular) interactions, which are also responsible for the non-ideal behaviour of real gases. On the other hand, Chemisorption is a type of adsorption whereby a molecule adheres to a surface through the formation of a chemical bond, as opposed to the Van der Waals forces which cause physisorption. Adsorption is usually described through isotherms, that is, functions which connect the amount of adsorbate on the adsorbent, with its pressure (if gas) or concentration (if liquid).. There are several models describing process of adsorption, namely Freundlich isotherm, Langmuir isotherm, BET isotherm, etc.
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2.7.1 Langmuir isotherm In 1916, Irving Langmuir published an isotherm for gases adsorbed on solids, which retained his name. It is an empirical isotherm derived from a proposed kinetic mechanism. It is based on four hypotheses: •
The surface of the adsorbent is uniform, that is, all the adsorption sites are equal.
•
Adsorbed molecules do not interact.
•
All adsorption occurs through the same mechanism.
•
At the maximum adsorption, only a monolayer is formed: molecules of adsorbate do not deposit on other, already adsorbed, molecules of adsorbate, only on the free surface of the adsorbent.
For liquids (adsorbate) adsorbed on solids (adsorbent), the Langmuir isotherm can be expressed by the equation -
M= Amax k.c 1+kc Activated carbon, also called activated charcoal, is a general term that includes carbon material mostly derived from charcoal. The three main physical carbon types are granular, powder and extruded (pellet). All three types of activated carbon can have properties tailored to the application. Activated carbon is frequently used in everyday life, in: industry, food production, medicine, pharmacy, military, etc. In pharmacy, activated charcoal is considered to be the most effective single agent available as an emergency decontaminant in the gastrointestinal tract. It is used after a person swallows or absorbs almost any toxic drug or chemical.
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Figure – 6 Langmuir isotherm 2.7.2 Biosorption sites During toxic waste product removal including pesticides, microbial cells play a dominant role. The cell wall is usually the first cellular structure that comes in contact with the chemical species in the extracellular environment. A microbial cell wall is a well-defined polymeric matrix located just outside the plasma membrane of a cell. Cell walls provide increased mechanical strength and resistance and can be composed of polysaccharides, proteins, lipids, or a combination of these compounds. They are part of the structure of eubacteria, archaebacteria, fungi, algae, and plants. Almost all eubacterial species have cell walls containing a polysaccharide called peptidoglycan. Among the eubacteria, differences in cell wall structure are a major feature used in classifying these organisms into two main groups: Gram Positive and Gram Negative bacteria. The cell walls of archaebacteria are distinctive from those of eubacteria being composed of different polysaccharides and proteins, with no peptidoglycan. Many archaebacteria have cell walls made of the polysaccharide pseudomurein. Fungal cell walls are typically composed of the polysaccharides chitin and cellulose, and the cell walls of algae and plants are composed mainly of the polysaccharide cellulose. Proteins, polysaccharides and nucleic acids are the three major polymer groups ubiquitous in the living world, being found in all members of the animal, plant, protozoan and microorganism groups. These biopolymers, constituents of the cell wall and the other parts of the cell possess functional groups that have a significant potential for chemical cationic and anionic species 19 | P a g e
binding. Furthermore, intracellular biopolymers such as proteins and DNA may also contribute to species immobilisation. As a generalization, the binding of chemical ions to biopolymers is likely to be via two major mechanisms, the first of these being simple ion-exchange and the second through the formation of complexes (co-ordination compounds) which may be chelates. Because of the complexity of most biopolymers, it is very likely that more than one processes of binding take place in a system at the same time. Two simplified examples are: - Ion exchange: via cation-binding with ionisable functional groups found in biopolymers such as carboxyl, organic phosphate and organic sulphate; Complex formation by organic molecules (ligands) with chemical entities: where one or more lone electron pairs of the ligand are donated to the parent chemical group. 2.7.3 Biosorption Mechanism The elucidation of the mechanism of biosorption is necessary to enable the technology to be developed. Such mechanisms are complicated and not fully understood. Several literature are cited concerned with the mechanism and modeling of biosorption referring to specific chemical groups and microbial strains. The key factors controlling and characterizing these mechanisms are - The type of biological ligands available for metal sequestering; the status of the biomass, i.e. living /non-living, the chemical, stereo chemical and coordination characteristics of the targeted metals and metal species and the characteristics of the metal solution such as pH and the presence of competing co-ions. Microorganisms possess an abundance of functional groups that can passively adsorb metal ions and chemical functional groups. The term adsorption can be used as a general term and includes several passive, i.e. non-metabolic, mechanisms such as: complexation; chelation; co-ordination; ion exchange; precipitation; reduction. The chemical functional group uptake by fungal biomass takes place by two basic processes. The first is by living organisms, where the chemical group uptake is dependent on the metabolic activity. The second process involves uptake by dead and living cells as a result of the chemical functional groups of the cell and, in particular, the cell wall. It should be noted that the chemical functional group uptake by the second process may also be involved during the metabolism-dependent metal uptake of growing cells (Gadd, 1986). 20 | P a g e
The cell wall of the fungi is the first to come into contact with functional group ions in solution, where the groups can be deposited on the surface or within the cell wall structure before interacting with the cytoplasmic material or the other cellular parts. In extreme cases, for the living cells, intracellular uptake may take place due to the increased permeability as a result of cell-wall rupture and subsequent exposure of the metal-binding sites (Gadd, 1990). The chemical species uptake by the cell wall has been broadly based on two mechanisms: uptake directed by functional groups like phosphate, carboxyl, amine and phosphate diester species of these compounds. The second uptake mechanism results from physicochemical inorganic interactions directed by adsorption phenomena. The removal mechanisms for radionuclides result from the combination of the above two processes, while for other heavy metals, the first process seems to play an important role. 2.7.4 Biosorption of pesticdes Application of biosorption for pesticides is also possible and several microorganisms including bacteria and fungi have been studied for the removal of some pesticides. The table represents a comparison of biosorption capacities of some microorganisms for some pesticides at their working conditions
pesticide
pH Temp(0C)
Activated sludge
Lindane
‐
20
Diazinon
‐
20
Malathion
‐
20
Bacillus subtilis
Lindane
‐
20
Bacillus megaterium
Lindane
‐
20
Emericella nidulns
2,4‐D
6.0
20
Escherichia coli
Lindane
‐
20
Mucor racemosus
PCNB
‐
21
Biosorbent
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Rhizopus arrhizus
PCNB
‐
21
Rhizopus arrhizus
Lindane
‐
20
Diazinon
‐
20
Malathion
‐
20
2‐Chlorobiphenyl
‐
20
PCNB
‐
21
Sporpthrix cyanescens
Table – 2 Data on biosorption of pesticides by various microorganisms 2.7.5 Microorganisms used in pesticide biosorption and biosorption mechanisms Bell and Tsezos (95) studied the biosorption of lindane, diazinon, malathion and 2chlorobiphenyl which are widely used organochlorine and organophosphorus insecticides, onto live and dead cells of activated sludge and R. arrhizus. They proposed that a part of the observed biosorptive uptake can be attributed to the cell walls of the microbial biomass. Moreover the biosorption process in the case of diazinon and lindane, involved an exothermic physical, rather than chemical, mechanism. They found that the octanol/water partition coefficient was directly related to the biosorptive uptake of these organics onto live and dead biomasses. Ju et al. (1997) investigated the biosorption of lindane, an organochlorine pesticide onto dried Gram-negative bacteria (E. coli, Zooglea ramigera) and Gram-positive bacteria (Bacillus megaterium, B. subtilis). They proposed that hydrophobic interaction and van der Waals forces are involved in the biosorption of lindane. They found that among the four bacteria, Z. ramigera showed the maximum uptake capacity.
Young and Banks (1998) used a heat treated non viable cell
suspension of the fungus R. oryzae for the removal of low concentrations of lindane from aqueous solution in a batch system. The results indicated that the mechanism of adsorption was by physical bonding of the negatively charged lindane molecule to the negatively charged fungal cell wall with hydrogen ions acting as the bridging ligand. Lievremont et al. (1998) reported on the removal of pentachloronitrobenzene (PCNB), a fungicide, from aqueous solution by dead fungal mycelia of Mucor racemosus, R. arrhizus, and 22 | P a g e
Sporothrix cyanescens and compared with sorption on isolated cell walls of these three strains. They proposed that biosorption involved both uptake by the cell walls and by other cellular components. Size of cells, morphology and chemical composition as well as the number of the active adsorption sites and their distribution may play a significant role in determining uptake capacity. Sorption of the adsorbate is also dependent on its molecular size and reactivity as well as mobility in the solution phase. Studies shown by Hong et al. (2000) studied the biosorption of 1,2,3,4-tetrachlorodibenzo- p-dioxin (1,2,3,4-TCDD) and some polychlorinated dibenzofurans (PCDFs) pesticides by Bacillus pumilus. The results showed that dead biomass of microorganism could remove these molecules from the medium more effectively than live cells. They suggested that in addition to the attachment to microorganisms itself, extracellular polymeric substances might also be involved in the biosorption process.
2.8 Effect of pH on pesticide biosorption Ju et al. (1997) investigated the effect of pH between 2.93 and 6.88 on the biosorption of lindane by E. coli, Z. ramigera, B. megaterium, and B. subtilis. They observed higher biosorption under lower pH. They found the isoelectric points of all bacteria at pH 2.0, except for E. coli whose isoelectric point is at 3.0 so all cells are negatively charged above these pH values. They proposed that the repulsive electrostatic force for the adsorption of organic halide on the cell surface decreases when a lower pH generates less negative charge on cell surfaces. As the cell and lindane molecules move closer to each other, owing to the decrease in electrostatic force, the van der Waals force is intensified and biosorption is enhanced consequently. Young and Banks (1998) studied at different pH values changing from 2.0 to 10.0 in order to investigate the effect of pH on the biosorption of lindane by heat treated R. oryzae and they found that biosorption was most effective at low pH.
2.9 Effect of initial pesticide concentration on pesticide biosorption The results obtained by Ju et al. (1997) indicated that the increase in initial lindane concentration from 1 to 4 mg l−1 at a constant cell concentration of 8 g l−1, increased the biosorption capacity of
23 | P a g e
lindane by dried bacteria of Z. ramigera, E. coli, B. subtilis, and B. megaterium from 370 to 2800 µg g−1, from 98 to 500 µg g−1, from 100 to 600 µg g−1, and 100 to 700 µg g−1.
2.10 Effect of ionic strength on pesticide biosorption Ju et al. (1997) investigated the effect of initial cell concentration on the biosorption of lindane by B. megaterium at an initial lindane concentration of 4mg l−1. The results obtained showed that lindane biosorption capacity increased from 340 to 700 mg g−1 with increasing cell concentration from 2 to 16g l−1. Young and Banks (1998) observed that the biosorption capacity of R. oryzae for lindane increased with increasing biomass density from 1 to 12 g l−1.
2.11 Equilibrium modeling and biosorption kinetics Bell and Tsezos (1987) applied the Freundlich model to the biosorption data of lindane, diazinon, malathion and 2-chlorobiphenyl on both activated sludge and R. arrhizus and they found that the isotherms for lindane were linear (1/n = 1). A large ultimate adsorption capacity and low energy of adsorption could account for the linear isotherm. For diazinon biosorption by R. arrhizus equilibrium data fitted the Freundlich model while the isotherm for activated sludge was essentially linear. Ju et al. (1997) defined the lindane biosorption equilibrium by E. coli, Z. ramigera, B. megaterium and B. subtilis in terms of the Freundlich model. Young and Banks (1998) also applied the Freundlich model successfully to the biosorption data of lindane on R. oryzae. Lievremont et al. (1998) also described the biosorption of PCNB on to dead fungal mycelia of M. racemosus, R. arrhizus, and S. cyanescens by the Freundlich model. Reports provided by Benoit et al. (1998) described the equilibrium data of 2,4-D by the pretreated E. nidulans by Freundlich model. It was observed that biosorption of lindane by E. coli, and other microorganisms at first was relatively rapid and slowed down later, reaching equilibrium within 4 h. According to Lievremont et al. (1998) found a contact time of 6 h was sufficient enough toattain equilibrium for the biosorption of PCNB on to dead fungal mycelia of M. racemosus, R.arrhizus, and S. cyanescens. The results obtained by Benoit et al. (1998) also indicated that a fast equilibrium between 2,4-D in solution and 2,4-D sorbed on pretreated E. nidulans was occurred within the first three hours of contact.
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The usage of inactive microorganisms for the removal of organics including dyes, phenolics and pesticides from wastewaters and the parameters affecting the biosorption rate and capacity has been reviewed in detail in this paper. Use of untreated (live) or treated (chemical or heat treated) microorganisms as biological adsorbents has attracted attention during recent years because of fastness, low cost, easy availability, easy operating conditions, high efficiency in detoxifying very dilute or concentrated effluents and no nutrient requirements and thus have been proposed to clean a variety of industrial effluents containing organic pollutants. Bacterial, fungal, and yeast strains are shown to be the main microorganism types capable of removing organics from soil sediments and wastewater. But the literature survey indicated that biosorption studies of pesticides in tropical Indian agricultural soils are very limited and only sorption of selected toxic organics onto a few types of bacterial, fungal and yeast biomass have been investigated. There is a need to study the pollutants related to chemical pesticides and to develop new strains which can be provided easily as waste and/or abundant biomass or can grow in simple, inexpensive medium and have high production rate and possess high biosorption capacity.
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Chapter – 3 Materials and Method 3.1 Collection, transport and processing of soil samples from agricultural sites Soil samples were collected both from rhizospheric and non rhizospheric regions of rice-rice (wet soil) cropping system in the farm of Orissa University of Agriculture and Technology (OUAT), Bhubaneswar and rice-sugarcane (dry soil) cropping system from agricultural fields of Nayagarh and Ganjam district of Odisha respectively. All soil samples were sampled as blocks and transported to the laboratory using sampling polythene bags. The samples were characterized as dry and wet and used for analysis accordingly. The soil samples are further enriched with supplementation of glucose at different concentrations. 3.2 Selected physico chemical properties of soil samples The physico-chemical characteristics of the soil in the present study, as presented in table, were determined using the following methods: • Soil pH was determined in 1:1.25 soil to water suspension using glass electrode pH meter (Jackson 1973). • Maximum water holding capacity (WHC) of soils was determined by the KeenRaczkowski method as outlined by Piper (1966). • Electrical conductivity (EC) was determined in supernatant of 1:1.25 soil to water suspension using conductivity bridge (Jackson 1973). • Organic carbon content of the soil samples was estimated by the Walkley-Black’s wetoxidation method as described by Jackson (1973). • Soil samples were analyzed for clay, silt, and sand fractions by employing Bouycous hydrometer method (Piper 1966).
3.3 Enrichment of soil samples using glucose as carbon source The soil samples in 25 g portions characterized as both wet and dry conditions were taken in 250 ml conical flask containing 50 ml of 0.9% of saline solution. To the contents of the flask, 5 gm of 26 | P a g e
glucose was supplemented and mixed uniformly. The flasks were put in a shaking incubator at 125 rpm for 12 hrs at 370C. After incubation, serial dilution was performed and plated using different enriched media. 3.4 Use of different culture media for cultivation of indigenous microbes Soil sample in 1 gm portions was taken in 25 ml falcon tube containing 9ml of autoclaved 0.9% saline solution. The soil suspension was vortex uniformly using vortex mixer. A series of dilution tubes were prepared from 10-1 to 10-8 by transferring 100 µl of soil suspension into each of the dilution tube containing 900 µl 0.9 % saline solution. Samples in 100 µl portions were spread uniformly on prepared media plates and were incubated for 24-48 hrs at 37° C. After incubation the microbial colonies obtained were pure cultured and characterized morphologically and biochemically.
3.4.1 Luria Bertani agar (LBA) medium Constituents Tryptone Yeast extract Sodium Chloride Agar powder PH Distilled water
Quantity in g/l 10 5 5 18 7.2 1L
3.4.2 Minimal LBA medium(X/2) Constituents Tryptone Yeast extract Sodium Chloride Agar powder PH Distilled water
Quantity in g/ml 0.5 0.25 0.25 1.8 7.2 100 ml
3.4.3 Minimal LBA medium(X/4) Constituents Tryptone 27 | P a g e
Quantity in g/ml 0.25
Yeast extract
0.125
Sodium Chloride
0.125
Agar powder 0
PH (at 37 C) Distilled water
1.8 7.2 100 ml
3.4.4 Soil extract agar medium (SEA) Constituents
Quantity in g/l
Glucose
1
Dipotassium phosphate (K2HPO4)
0.5
Soil extract
17.75
Agar
15
PH (at 25°C)
6.8±0.2
10 gm of soil sample in 10 gm portions were taken in 50 ml falcon tube containing 20 ml of distilled water was added. The suspension was mixed uniformly and centrifuged at 2000 rpm To the constituents distilled water was added to make up the volume to 1 liter. The final PH was adjusted properly and agar powder was added and stirred properly.
3.4.5 Glucose agar medium (GAM) Constituents
Quantity in g/l
Glucose
10
Peptone
2
Sodium Chloride
5
Dipotassium phosphate (K2HPO4)
0.3
Bromothimol blue
15 ml
Agar
18
PH (at 25°C)
6.8±0.2
Distilled water
28 | P a g e
1L
3.4.6 Potato Dextrose Agar (PDA) PDA Suspended 39 gms in 1000 ml distilled water. It was heated and boiled to dissolve the medium completely. It was sterilized by autoclaving. It was mixed well before dispensing. Potatoes Constituents
Quantity in g/l
Potatoes, infusion from
200
Dextrose
20
Agar
15
Final pH ( at 25°C)
5.6±0.2
3.4.7 Czapek dox Agar: Constituents
Quantity in g/l
Sucrose
30
Sodium nitrate (NaNO3)
3
Magnesium sulfate (MgSO4)
0.5
Ferrous sulphate (FeSO4)
0.5
Di-potassium hydrogen phosphate ( K2HPO4)
1
Yeast extract
5
Agar
17
PH
7.3±0.2
Supplementation of yeast extract (5g/l) and PH value of 4.0 for growth of soil dwelling fungus. Bacterial can be inhibited by adding 30 mg/l Streptomycin inoculate by spreading the sample thinly on the surface of prepared plates and incubated aerobically at 280C.
3.5 Cultivation of Fungal biomass : For cultivation of fungal biomass from collected soil sample with and without glucose supplementation, 250 ml of Potato Dextrose Broth and Czapek-Dox Broth was prepared and autoclaved. To the contents of the flask, 5 ml of soil suspension and streptomycin (1mg ml-1) was added. The conical flasks were incubated in a shaker at 140 rpm for 5-6 days at 280C. Following incubation period, the contents of the flask were centrifuged at 10,000 rpm for 10 mins, and 29 | P a g e
filtered using Whatman’s filter paper. The filtrate was oven dried at 600 C for 24 hours and crushed into fine powder using mortar and pestle. The obtained biomass was stored using sterile containers for biosorption studies.
3.6 Biochemical characterization Biochemical characterization was mainly conducted for identification of microorganisms. The tests were performed to indicate the different characteristics of microorganism with respect to subjected bio chemicals which included primarily cell shape (microscopic), motility, Catalase test , MR-VP test, Simmon’s citrate test, Amylase test.
3.7 Reduction potential assay of pesticides For determining the reduction potential assay for pesticides, Chlorpyrifos (coded as A), Monocrotophos (coded as B), Malathion (coded as C) and Dicofol (coded as D) were used. Selected cultures were swabbed on LBA and minimal media plates respectively. Using sterile microtips, the swabbed plates were punctured to make uniform wells at 4 different quadrants. Serial dilutions of pesticides A, B, C, D were done from 10-1 to 10-4 respectively. About 40µl of diluted pesticide samples were inoculated in the wells against each definite dilution previously marked on the plate. The plates were incubated in an upright position at 370 C overnight. The results were further confirmed by performing Paper disc diffusion assay to determine the zone of inhibition.
3.8 Effect of adsorbate on adsorption of pesticides in soil samples To determine the sorption of pesticides using adsorbate such as water melon seeds, about 100 mg of dried watermelon seeds (crushed) were taken in 250 ml of conical flask containing 100 ml of distilled water was mixed. The conical flask was put in the shaker at 140 rpm overnight. The flask was divided into two equal sets of 50 ml each for with and without heat treatment. For blank 100 mg of dried watermelon seeds (crushed) were taken in 250 ml of conical flask containing only distilled water
30 | P a g e
3.8.1 Heat and without treatment Selected pesticide such as Monocrotophos was added to 250 ml conical flask containing
50
ml sample each. One of the flask was incubated at room temperature for about 30-45 mins and the second flask was heated at 80 °C for 20 mins. Then the flasks were transferred to the shaking condition having 140 rpm. At an interval of 15 mins, 1 ml of the sample was centrifuged and the absorbance peak value was measured spectrophotometrically at different wavelengths. The experiment was repeated up to 90 mins and the data were recorded for analysis. . 3.9 Effect of sorption of pesticides by isolated fungal biomass in soil samples Selected pesticide such as Monocrotophos, Malathion, Chlorpyrifos and Dicofol was added to 250 ml conical flask containing 50 ml sterile distilled water and 100 mg each of isolated powdered fungal biomass. All the flasks were incubated at room temperature for about 30-45 mins under shaking condition at 140 rpm. At an interval of 15 mins, about1 ml of the sample was centrifuged and the absorbance peak value was measured spectrophotometrically at selected wavelengths of ( 226 nm, 272 nm, 280 nm and 277 nm ) The experiment was repeated up to 90 mins and the data were recorded for analysis.
3.10 Effect on sorption of pesticides by isolated mixed fungal biomass in soil samples Selected pesticide such as Monocrotophos, Malathion, Chlorpyrifos and Dicofol was added to 250 ml conical flask containing 50 ml sterile distilled water and isolated powdered fungal biomass at different proportions of 1:1, 1:2 and 2:1 respectively. All the flasks were incubated at room temperature for about 30-45 mins under shaking condition at 140 rpm. At an interval of 15 mins, about1 ml of the sample was centrifuged and the absorbance peak value was measured spectrophotometrically at selected wavelengths of (226 nm, 272 nm, 280 nm and 277 nm) .The experiment was repeated up to 90 mins and the data were recorded for analysis.
3.11 Effect of pH on sorption of pesticides by isolated mixed fungal biomass in soil samples To examine the effect of pH on sorption process, selected pesticide such as Monocrotophos, Malathion, Chlorpyrifos and Dicofol was added to 250 ml conical flask containing 50 ml sterile 31 | P a g e
distilled water and isolated powdered fungal biomass at proportions of 1:1 respectively. The samples were then set at different pH of 3, 5, 7 and 9 respectively All the flasks were incubated at room temperature for about 30-45 mins under shaking condition at 140 rpm. At an interval of 15 mins, about1 ml of the sample was centrifuged and the absorbance peak value was measured spectrophotometrically at selected wavelengths of (226 nm, 272 nm, 280 nm and 277 nm) The experiment was repeated up to 90 mins and the data were recorded for analysis.
3.12 Effect of agitation speed on sorption of pesticides by isolated mixed fungal biomass in soil samples To examine the effect of agitation speed of samples on sorption process, selected pesticide such as Monocrotophos, Malathion, Chlorpyrifos and Dicofol was added to 250 ml conical flask containing 50 ml sterile distilled water and isolated powdered fungal biomass at proportions of 1:1 respectively. The samples were then agitated at 75, 125, 150 and 200 rpm respectively. At an interval of 15 mins, about1 ml of the sample was centrifuged and the absorbance peak value was measured spectrophotometrically at selected wavelengths of (226 nm, 272 nm, 280 nm and 277 nm) The experiment was repeated up to 90 mins and the data were recorded for analysis.
3.13 Statistical analysis All analyses were carried out on the three replicates. The data were analyzed statistically by the analysis of variance (ANOVA) procedure using Origin8 statistical softyware.
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Chapter – 4 Results and Discussion The soil samples after collection both from rhizospheric and non rhizospheric regions of rice-rice (wet soil) cropping system in the farm of Orissa University of Agriculture and Technology (OUAT), Bhubaneswar and rice-sugarcane (dry soil) cropping system from agricultural fields of Nayagarh and Ganjam district of Odisha were analyzed for different physico-chemical properties such as pH, electrical conductivity, organic carbon content, maximum water holding capacity and soil fractions including proportions of sand silt and clay (Table -1). The soil samples varied in water holding capacity ranging from 32.45 % to 45.68% as observed in alluvial soil of agricultural fields of Ganjam and Nayagarh respectively. On the other hand, it was evident that the organic content represented as 0.75% was significantly least among all the soil samples. All the soil samples have a pH range between 5.89 to 6.62 respectively. The proportions of sand, silt and clay were however similar in alluvial soil of Nayagarh and laterite soil of Bhubaneswar the samples. ________________________________________________________________________ Soil Coding pH EC Organic WHC Clay Silt Sand (ds m‐1) C (%) (%) _______________________________________________________________________________ Laterite‐ BBSR (L‐BBSR) 6.62 0.79 1.12 43.70
32.00 27.00 41.00
Alluvial‐ NYG (A‐NYG) 6.05 0.65 1.76 45.68
34.00 29.00 37.00
Alluvial‐ GJM (A‐GJM) 5.89 0.85 0.75 32.45
19.00 11.20 79.80
________________________________________________________________________________
Table 3. Physico‐chemical properties of different soils used in the present study
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4.2 Soil enrichment and use of different culture media for the cultivation of indigenous microorganisms The enrichment of soil samples with glucose as carbon source and the use of basic microbiological techniques for the cultivation of indigenous microbes in different media including Luria bertani agar, minimal media, glucose agar media, soil extract medium, potato dextrose agar and czapek dox agar showed growth of bacterial and fungal colonies which were
Figure – 7 Cultivation and isolation of microbes using microbial techniques from soil samples 34 | P a g e
pure cultured and subsequenly characterized as shown in the Table -2. All microbial cultures were characterized as bacterial and fungal respectively. The obtained microbial cultures were inoculated in specific media for different biochemical tests including catalase test , MR-VP test, simmon’s citrate test, amylase test. Sl no
1
Sample source
OUAT - BBSR
Medium
Dilution
Morphology
Coding
LB agar
10-3
Transparent; small
A1
sized; slimy; creamy coloured 2
OUAT- BBSR
LB agar
10-3
Small shaped;
A2
dotted; Orange coloured 3
OUAT - BBSR
LB agar
10-3
Round shaped;
A3
slightly elevated; medium sized; whitish coloured 4
OUAT - BBSR
LB agar
10-3
Arbitrary shaped;
A4
Slightly bulged; creamy coloured 5
OUAT - BBSR
LB agar
10-3
Round shaped;
A5
rough margin; medium sized; creamy coloured 6
OUAT - BBSR
LB agar
10-3
Transparent; ununiform margin; not so dense;
35 | P a g e
A6
creamy coloured 7
OUAT - BBSR
LB agar
10-3
Small sized; round
A7
shaped; pure white coloured 8
OUAT - BBSR
LB agar
10-4
Arbitrary shaped;
A8
slimy; creamy coloured 9
OUAT - BBSR
LB agar
10-3
Big shaped;
A9
elevated; non uniform margin; creamy coloured 10
OUAT - BBSR
LB agar
10-4
Small sized; round
A10
shaped; white coloured but yellowish at centre 11
OUAT - BBSR
LB agar
10-3
Round shaped;
A11
medium sized; yellowish coloured 12
OUAT - BBSR
LB agar
10-4
Arbitrary shaped;
A12
non uniformed margin; slightly bulged; creamy coloured 13
Agricultural dry soil+Glucose; Nayagarh
36 | P a g e
LB agar
10-3
Small sized; irregular mat; dense; yellowish
A13
coloured 14
Agricultural dry
LB agar
10-3
soil+Glucose;
Agricultural dry
A14
irregular mat; pale
Nayagarh 15
Mesh like fibres; white coloured
LB agar
10-3
soil+Glucose;
Round; small; dry;
A15
elevated; complete
OUAT
margin; translucent; white coloured
16
Agricultural dry
LB agar
10-3
Round; opaque;
soil+Glucose;
complete margin;
Nayagarh
irregular shaped;
A16
pale white coloured 17
Agricultural wet
LB agar
10-2
soil+Glucose;
Irregular mat;
A17
spreaded largely;
OUAT
non uniform margin; pale white coloured
18
Agricultural wet
LB agar
10-2
soil+Glucose;
Agricultural wet soil+Glucose; OUAT
colour LB agar
10-2
Small; uniform margin; round; elevated surface; yellowish coloured
37 | P a g e
A18
round; orange in
OUAT 19
Small shaped;
A19
20
Agricultural wet
LB agar
10-2
soil+Glucose;
Agricultural dry soil+Glucose; OUAT
A20
uniform margin;
Nayagarh 21
Round shaped; yellowish coloured
Potato
Using soil
Mesh like
dextrose agar
suspension
translucent; non
(PDA)
A21
uniform margin; slimy; yellowish coloured
22
Agricultural wet soil+Glucose; Nayagarh
Potato
Using soil
Small shaped;
dextrose agar
suspension
translucent; dotted;
(PDA)
A22
pale white coloured
23
Agricultural wet soil+Glucose; OUAT
Potato
Using soil
Mesh like
dextrose agar
suspension
structure;
(PDA)
A23
translucent; non uniform margin
24
Agricultural dry soil+Glucose; Nayagarh
Potato
Using soil
Round shaped;
dextrose agar
suspension
small; uniform
(PDA)
margin; pale white coloured
38 | P a g e
A24
25
Agricultural dry soil+Glucose; Nayagarh
26
Agricultural wet soil; Nayagarh
Potato
Using soil
Round feathery
dextrose agar
suspension
structure; Black in
(PDA)
A25
colour
Glucose agar
Using soil
Round shaped,
medium
suspension
small; translucent;
(GAM)
A26
yellowish surface and orange dot at center
27
Agricultural wet soil; Nayagarh
Glucose agar
Using soil
Round shaped,
medium
suspension
very small;
(GAM)
A27
translucent; orange coloured
28
Agricultural dry soil; Nayagarh
Glucose agar
Using soil
Round; feathery
medium
suspension
margin; dry center;
(GAM) 29
Agricultural dry soil; Nayagarh
white in colour
Glucose agar
Using soil
Small; round; semi
medium
suspension
dry; yellowish
(GAM) 30
Agricultural wet soil; OUAT
A28
A29
coloured
Glucose agar
Using soil
Circular; small;
medium
suspension
dry; media
(GAM)
A30
embedded; pale yellow in colour
31
Agricultural wet soil; Nayagarh
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Glucose agar
Using soil
Circular; dry;
medium
suspension
media embedded;
A31
(GAM) Agricultural dry soil; OUAT
32
yellow in colour
Glucose agar
Using soil
Irregular mat; dry
medium
suspension
margin;
(GAM)
A32
translucent; white in colour
Table ‐4 Morphologically characteristics of isolated microbial samples
4.3 Biochemical characterization: •
Biochemical characterization studies are generally conducted for identification of microorganisms. The test indicates different characteristics of microorganism with respect to subjected bio-chemicals aiding identification up to species level. These tests are done under aseptic conditions under totally controlled environments in order to avoid un-wanted contamination and to keep the validity of test. In catalase test, oxygen are used as final electron acceptor by many micro-organisms the breakdown of H2O2 into water and oxygen molecules is characterized by bubble formation and this indicates the result as Positive (+). MRVP test, determines if organic acid is produced. Simmon’s citrate test identifies the ability of microbes to ferment citrate. In this test when citric acid or sodium citrate is in solution, it loses sodium ion to form citrate ion enabling the medium to turn alkaline.
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Figure ‐8 The figure A, B, C indicates the biochemical tests for the isolated microorganisms from collected soil samples
Biochemical analysis Organism
citrate
MR
VP
Catalase
Amylase
A4
++
++
-
-
-
A5
++
+++
-
+
-
A8
++
+++
-
-
-
A12
++
++
-
-
-
A18
+++
+++
-
+
-
A19
+++
+++
-
+
-
A31
+++
++
-
+
+
Table ‐4 Morphologically characteristics of isolated microbial samples
4.4 Growth Kinetics of isolated microbial cultures The isolated organisms A4, A5, A8, A12, A18, A19, A31 show a typical growth curve pattern with lag phase, log phase and stationary phase respectively. All the isolated cultures had a 41 | P a g e
significant log phase between 20 hrs to 80 hrs and a stationary phase till 120 hrs. There was a sharp decline in the microbial growth indicating declining phase.
4.5 Reduction potential assay of pesticides In order to determine the reduction potential assay for selected pesticides, Chlorpyrifos (coded as A), Monocrotophos (coded as B), Malathion (coded as C) and Dicofol (coded as D) were used. Selected cultures having high cell growth were swabbed on LBA and minimal media plates respectively. Using sterile microtips, the swabbed plates were punctured to make uniform wells at 4 different quadrants. Serial dilutions of pesticides A, B, C, D were done from 10-1 to 10-4 respectively.
Figure – 9 reduction potential assay of pesticides by disc diffusion assay
As the three cultures (A3, A4, A5) give positive result, we again swabbed the 3 organisms on LB agar plate to know the proper dilution which is responsible for zone of inhibition by well diffusion method.
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4.6 Effect of adsorbate on adsorption of pesticides in soil samples 80 y=a+(b-a)xn/(kn+xn)
Adsorbtion efficiency
70 60 50 40
y=a+(b-a)xn/(kn+xn)
30 20
Experimental data Hill 1 fitted curve for (a) Hill 1 fitted curve for (b)
10 0
0
10
20
30
40
50
60
70
Time (min)
Figure -10 Adsorption of pesticides under heat and without heat treatment
Determination of adsorption kinetics of the pesticide Monocrotophos, under both heat treatment and without heat treatment condition was carried out spectrophotometrically. The black dots representing without heat treatment shows better adsorption potential as compared to with heat treatment. The adsorption potential was maximum between 30-40 mins followed by desorption phenomena. The application of heat provides an increase in temperature which opens the pore size of the adsorbate and results in the deformity of the active site. 4.7 Kinetic sorption study of microbial biomass
80
Adsorbtion efficiency
70 60 50 40 30 Experimental data Hill 1 fitted curve for (a) Hill 1 fitted curve for (b) Hill 1 fitted curve for (c)
20 10 0
0
10
20
30 40 50 Time (min)
60
70
80
Figure -11 Kinetic biosorption studies of different pesticides
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Four numbers of pesticides were used for determining the kinetics of adsorption. The adsorption rate was maximum in chloropyrifos and least in dicofol. The mobility of the pesticide molecules is dependent upon the size of the functional group. The greater the size, lesser the mobility and more the adsorption 4.8 Effect of factors such as pH, agitation time on biosorption of pesticides
100
Experimental data Hill 1 fitted curve for (a) Hill 1 fitted curve for (b) Hill 1 fitted curve for (c) Hill 1 fitted curve for (d)
80
Adsorbtion efficiency
Adsorbtion efficiency
100
60 40 20 0
0
10
20
30 Time (min)
40
50
80 60 40
0
60
Experimental data Hill 1 fitted curve for (a) Hill 1 fitted curve for (b) Hill 1 fitted curve for (c) Hill 1 fitted curve for (d)
20
0
10
20
30 40 Time (min)
50
60
70
Figure -12 Kinetic biosorption studies of different pesticides at different pH
To examine the effect of pH on sorption process, selected pesticide such as Monocrotophos, Malathion, Chlorpyrifos and Dicofol was added to 250 ml conical flask containing 50 ml sterile distilled water and isolated powdered fungal biomass at proportions of 1:1 respectively. The samples were then set at different pH of 3, 5, 7 and 9 respectively Decrease in pH causes a change in molecular structure, so decreases kinetics of adsorption potential Similar trends was also observed for malathion whose adsorption potential was maximum at pH 9 To examine the effect of agitation speed of samples on sorption process, selected pesticide such as Monocrotophos, Malathion, Chlorpyrifos and Dicofol was added to 250 ml conical flask containing 50 ml sterile distilled water and isolated powdered fungal biomass at proportions of 1:1 respectively. The samples were then agitated at 75, 125, 150 and 200 rpm respectively. At an interval of 15 mins, about1 ml of the sample was centrifuged and the absorbance peak value was measured spectrophotometrically at selected wavelengths of (226 nm, 272 nm, 280 nm and 277 nm) It was observed that increase in agitation speed enhances the bio sorption efficiency 44 | P a g e
100
80
(a)
Adsorbtion efficiency
Adsorbtion efficiency
100
60 (b)
40
(c) (a) Experimental data Hill 1 fitted curve for (a) Hill 1 fitted curve for (b) Hill 1 fitted curve for (c) Hill 1 fitted curve for (d)
20 0
0
10
20
30 40 Time (min)
50
60
80 60 40
0
70
Experimental data Hill 1 fitted curve for (a) Hill 1 fitted curve for (b) Hill 1 fitted curve for (c) Hill 1 fitted curve for (d)
20
0
10
20
30 40 Time (min)
50
60
70
Figure -13 Kinetic biosorption studies of different pesticides at different agitation speed
CONCLUSION •
In the present study a diversity of microorganism were isolated using different basic and enriched medium. A total number of 32 isolates were screened, out of which 7 microbial cultures having higher biomass were selected for adsorption study
•
The rate of adsorption was significantly higher in isolated biomasses under without heat treatment condition. Whereas, with heat treatment adsorption efficiency was significantly less.
•
Factors such as Ph and agitation speed do play a key role in adsorption efficiency. Results confirmed that increase in Ph and agitation speed enhances the adsorption efficiency of pesticides by the isolated microbial biomass.
•
The kinetics of above adsorption studies follows the pattern of Hill’s equation.
FUTURE PROSPECTS •
Experimental analysis suggests the use of microbial biomass in biosorption of chemical pesticides do depend upon other abiotic factors which may play a key role in making bioremediation strategies at contaminated sites.
•
Application at chemical contaminated sites requires process optimization of adsorptiondesorption phenomena and the use of bench-scale bioreactors
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