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Azotobacter—A Natural Resource for Bioremediation of Toxic Pesticides in Soil Ecosystems
C H A P T E R
19 Azotobacter—A Natural Resource for Bioremediation of Toxic Pesticides in Soil Ecosystems G. Chennappa*, Nidoni Udaykumar*, M. Vidya†, H. Nagaraja‡, Y.S. Amaresh§, and M.Y. Sreenivasa‡ *Department of Processing and Food Engineering, College of Agricultural Engineering, Raichur, India † Centre for Nanotechnology, Department of Processing and Food Engineering, Raichur, India ‡ Department of Studies in Microbiology, University of Mysore, Mysore, India § Department of Plant Pathology, College of Agriculture, Raichur, India
O U T L I N E 19.1 Introduction
267
19.8 Effect of Pesticides on Natural Biodiversity
272
19.2 Benefits of PGPR
268
19.9 Effect of Insecticides on IAA Production
272
19.3 Plant Growth-Promoting Substances 19.3.1 Vitamins 19.3.2 Amino Acids 19.3.3 Indole Acetic Acid 19.3.4 Gibberellic Acid 19.3.5 Phosphate Solubilization 19.3.6 Natural Biocontrolling Agents 19.3.7 Hydrogen Cyanide 19.3.8 Siderophores
269 269 269 269 269 269 270 270 270
19.10 Effect of Insecticide on Nitrogen Fixation
272
19.11 Effect of Insecticides on GA Production
274
19.12 Effect of Insecticide on Phosphate Solubilization
275
19.13 Biodegradation of Pesticides
275
19.4 Effect of Pesticides
270
19.15 Conclusions
277
19.5 Impact of Pesticides on Environment
271
References
277
19.6 Impact of Pesticides on Soil and Water
272
19.7 Impact of Pesticides on Human Beings
272
19.14 Biodegradation of Insecticides by Azotobacter Species 275
19.1 INTRODUCTION Plant protection has been an integral part of agricultural practices and average yield losses in India are estimated to be 10%–30% which is caused by insects, diseases, and weeds. Nearly a total of 43.5% pesticides are used to protect cotton and 38.6% for protection of rice crop (Kadam and Gangawane, 2005). Presently different types of formulated pesticides are used worldwide to control wide spread of diseases and to minimize the economic losses
New and Future Developments in Microbial Biotechnology and Bioengineering https://doi.org/10.1016/B978-0-444-64191-5.00019-5
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© 2019 Elsevier B.V. All rights reserved.
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19. AZOTOBACTER—A NATURAL RESOURCE
TABLE 19.1
Use of Different Types of Pesticides in Agriculture and Their Target of Diseases
Sl. no.
Pesticides
Chemical nature
Target hosts
1
Pendimethalin
Herbicides
Broad leaves, Grass
2
Phorate
Nematodes
Hershieminella oryzae
3
Glyphosate
Herbicides
Cyanodondoctylon
4
Simazine
Herbicides
Grass plants
5
Malathion
Insecticides
Leaf hoppers
6
Carbendazim
Fungicides
Pericularia oryzae
7
Toxaphene
Insecticide
Fruit borer
8
Carbofuran
Nematodes
Hershieminella oryzae
9
Monocrotophos
Insecticide
Brown plant hopper
10
Endosulfan
Insecticide
Fruit and leaf borer
11
Chloropyrifos
Insecticide
Yellow stem borer
12
Methyl parathion
Insecticide
Stem and leaf borer
(Chowdhary et al., 2018; Bharagava and Mishra, 2018). Unscientific and inappropriate use of these pesticides may diminish the population of beneficial microorganisms present in the soil. Among them, chemical pesticides and chemical fertilizer reaching the soil in significant quantities have direct effect on soil microbiological aspects, environmental pollution, and health hazards. Due to its excessive use and its distribution in the environment it pollutes soil, water bodies, and enters the food chain causing neurotoxicological disorders in animals, birds, and human beings (Martin et al., 2011). Pesticides cause everlasting changes in the soil microflora (Aleem et al., 2003), adverse effect on soil fertility and crop productivity, inhibition of nitrogen-fixing bacteria (Sachin, 2009), interference with ammonification, adverse effect on mycorrhizal symbiosis in plants and nodulation in legumes (Reinhardt et al., 2008). Pesticide concentration alters the soil ecosystem, soil microbe interaction, plant growth and soil structure, organic matter decomposition, biogeochemical cycling of elements. Several toxic and carcinogenic pesticides have been banned in many of the countries including India. Number of microbial species has the ability to break down toxic into simpler nontoxic compounds (Chowdhary et al., 2018; Bharagava and Mishra, 2018). To overcome pesticide effects, several biodegradation works have been carried out in order to minimize the pesticides residues in food and feed. For biodegradation of toxic pesticides, several types of soil-borne bacteria and fungi are widely used such as species of Arthrobacter, Burkholderia, Bacillus, Azotobacter, Flavobacteria, Trichoderma, Pseudomonas, and Rhodococcus (Chennappa et al., 2015; Castillo et al., 2011; Kadam and Gangawane, 2005; Moneke et al., 2010; Bagyaraj and Patil, 1975; Ramaswami et al., 1977). The application of different pesticides against the target host is presented in Table 19.1. Among the PGPR group, genus Azotobacter has the potentiality to produce different types of plant growthpromoting substances such as amino acids, plant growth hormones, antifungal antibiotics, and siderophore and fixes atmospheric-free nitrogen in soil (Chennappa et al., 2013, 2014, 2016; Myresiotis et al., 2012). Azotobacter species happens to be the most dominant species in the rhizosphere soil and can biodegrade different types of pesticide and substituted phenolic compounds used for the management of plant diseases (Li et al., 1991). Hence, degradation of such pollutant plays an important role in agricultural practice to reduce the load of pesticides from the soil and to serve safe food to the world and clean environment for future generation (Sujata and Bharagava, 2016; Kumari et al., 2016; Goutam et al., 2018).
19.2 BENEFITS OF PGPR Plant growth-promoting rhizobacteria (PGPR) are known to produce different types of secondary metabolites under congenial conditions. These metabolites include vitamins, amino acids, plant growth hormones, antifungal metabolites, hydrogen cyanide (HCN), and siderophores. These plant growth-promoting substances have direct influence on overall plant growth of several agricultural crops (Chennappa et al., 2018a; Myresiotis et al., 2012; Ahmad et al., 2005).
19.3 PLANT GROWTH-PROMOTING SUBSTANCES
269
19.3 PLANT GROWTH-PROMOTING SUBSTANCES 19.3.1 Vitamins PGPR group are one of the beneficial bacterial genera and Azotobacter species are one of the important bacterial group of PGPR’s known to produce different types of vitamins under favorable conditions. Azotobacter vinelandii (ATCC 12837) and Azotobacter chroococcum (CECT 4435) strains produced B-group vitamins which include niacin (Vit B3), pantothenic acid, riboflavin, and biotin. Vitamins are essential compounds for the physiological functions of the living beings which are produced by several groups of bacteria (Revillas et al., 2000). Riboflavin is a vitamin B2 required for a wide variety of cellular processes and it plays a key role in metabolism of fats, ketone bodies, carbohydrates, and proteins, respectively. Genetically engineered Bacillus subtilis and Corynebacterium ammoniagenes are used for mass production of riboflavin by which they overexpress genes of the enzymes involved in riboflavin biosynthesis (Almon, 1958; Revillas et al., 2000).
19.3.2 Amino Acids Amino acids are also important components for all living beings. Amino acids are produced by various processes and different types of microbes are also able to produce amino acids. Species of Azotobacter species produces different types of amino acids under diazotrophic conditions whereas growth media amended with glucose as sole carbon source (Lopez et al., 1981). Species of A. vinelandii and A. chroococcum are known to produce aspartic acid, serine, glutamic acid, glycine, histidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine (Revillas et al., 2000).
19.3.3 Indole Acetic Acid Indole acetic acid (IAA) is important plant auxin produced by different groups of soil-borne bacteria commonly living in plant rhizosphere (Barazani and Friedman, 1999). The IAA synthesizing ability has been detected in many rhizobacterial species as well as in pathogenic, symbiotic, and free-living bacterial species. Saline soil is a rich source of IAA-producing bacteria with 75% of the bacterial isolates is reported to be active in IAA production. Azotobacter species are found to produce IAA in the range of 2.09–33.28 μg/mL (Spaepen et al., 2007; Chennappa et al., 2016). Most commonly, IAA-producing PGPR strains are known to increase root length resulting in greater root surface area which enables plants to access more nutrients from soil. The IAA is responsible for the division, expansion, and differentiation of plant cells, tissues, and stimulates root elongation (Ahmad et al., 2008). Among PGPR species, Azospirillum, Aeromonas, Burkholderia, Azotobacter, Bacillus, Enterobacter, Pseudomonas, and Rhizobium are the most studied IAA-producing bacterial species which have been isolated from different rhizosphere soils (Reshma et al., 2018; Ahmad et al., 2008; Ghosh et al., 2010).
19.3.4 Gibberellic Acid Another important auxin, that is, gibberellins produced by several types of bacteria which include a wide range of chemicals that are produced naturally within plant rhizosphere. Gibberellic Acid (GA) produced by fungi called Gibberella fujikuroi in rice plant was first discovered and reported by Japanese scientist Eiichi Kurosawa. Gibberellins are important for seed germination, enzyme production that mobilizes growth of new cells. GA promotes flowering, cellular division and seed growth (Upadhyay et al., 2009).
19.3.5 Phosphate Solubilization Phosphobacteria play a significant role in the biotransformation of phosphate to soluble phosphorous form and are capable of hydrolyzing organic and inorganic phosphorus from insoluble compounds. Species of A. chroococcum, B. subtilis, B. cereus, B. megaterium, Arthrobacter ilicis, E. coli, P. aeruginosa, E. aerogenes, and Micrococcus luteus were identified as phosphate-solubilizing bacteria (Garg et al., 2001; Kumar et al., 2000). Chennappa (2016) reported the maximum level of phosphate-solubilizing Azotobacter species.
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19. AZOTOBACTER—A NATURAL RESOURCE
FIG. 19.1 Various plant growth-promoting attribute of Azotobacter sp.
19.3.6 Natural Biocontrolling Agents The production of antibiotics is considered as one of the most studied biocontrol mechanisms for combating phytopathogens. Azotobacter produces antifungal antibiotic compounds (2, 3-dihydroxybenzoic acid, aminochelin, azotochelin, protochelin, and azotobactin) which inhibit the growth of plant pathogenic fungi, viz., Aspergillus, Fusarium, Curvularia, Alternaria, and Helminthosporium (Nagaraja et al., 2016; Khan et al., 2008; Mali and Bodhankar, 2009; Kraepiel et al., 2009). The 2, 4-DAPG is one of the most efficient antibiotics produced by Pseudomonas and it has a wide spectrum of antifungal, antibacterial, and anthelmintic properties (Naik et al., 2013). Azotobacter species act as biocontrol agents for many plant pathogens and Azotobacter armeniacus inhibited growth of Fusarium verticillioides (Mali and Bodhankar, 2009; Agarwal and Singh, 2002).
19.3.7 Hydrogen Cyanide Species of Azotobacter, Alcaligenes, Aeromonas, Bacillus, Pseudomonas, and Rhizobium capable of producing HCN a volatile, secondary metabolite that suppresses the growth of microorganisms and also influences the growth and development of plants (Ahmad et al., 2008; Chennappa et al., 2018a, b).
19.3.8 Siderophores Siderophores are produced and utilized by bacteria and fungi as iron (Fe)-chelating agents which are produced in response to iron deficiency which normally occurs in neutral to alkaline pH soils ( Johri et al., 2003). Azotobacter excretes siderophores under limited iron conditions and A. vinelandii produced five different siderophores, viz., 2, 3-dihydroxybenzoic acid, aminochelin, azotochelin, protochelin, and azotobactin. The main applications of siderophores act as drug delivery agents, antimicrobial agents, and soil remediation (Mollmann et al., 2009; Kraepiel et al., 2009; Barrera and Soto, 2010; Page and Von Tigerstrom, 1988). Azotobacter species also produces poly-bhydroxybutyrate (intracellular polyester), alginate, and catechol (extracellular polysaccharide) compounds under favorable environmental conditions (Barrera and Soto, 2010). PHB are used in large-scale production of biodegradable and biocompatible thermoplastic used as substitute for polyethylene and polypropylene. All the above beneficial aspects of Azotobacter species has been depicted in Fig. 19.1.
19.4 EFFECT OF PESTICIDES To control pests and diseases, farmers are applying excess quantity of pesticides and chemical fertilizers as compared to the recommended dosages. Several pesticides are regularly applied for the cultivation, viz., pendimethalin, phorate, glyphosate, simazine, malathion, carbofuran, monocrotophos, diazinon, fenthion, phosphamidon, methyl parathion,
271
19.5 IMPACT OF PESTICIDES ON ENVIRONMENT
TABLE 19.2 Degradation of Pesticides by Different Bacterial Species Sl. no.
Bacteria
Pesticides
References
1
Micrococcus
Chloropyrifos
Guha et al. (1997)
2
Flavobacterium sp.
Glyphosate
Singh and Walker (2006)
3
Enterobacter strain B-14
Chloropyrifos
Singh and Walker (2006)
4
Alcaligenes faecalis
Glyphosate
Singh and Walker (2006)
5
Rhodococcus erythropolis sp.
Carbendazim
Zhang et al. (2013)
6
Klebsiella sp.
Chloropyrifos
Ghanem et al. (2007)
7
Pseudomonas sp.
Monocrotophos
Singh and Walker (2006)
8
Bacillus pumillus strain C2A1
Chloropyrifos
Anwar et al. (2009)
9
Lactobacillus bulgaris
Chloropyrifos
George et al. (2014)
10
Serratia sp.
Chloropyrifos
Gangming et al. (2007)
11
Agrobacterium sp.
Chloropyrifos
George et al. (2014)
12
Azotobacter chroococcum
Endosulfan
Castillo et al. (2011)
13
Bacillus circulans
Pendimethalin
Megadi et al. (2010)
14
A. chroococcum
Phorate
Kadam and Gangawane (2005)
chlorpyrifos, carbendazim, mancozeb, and azinophos methyl, etc. (Chennappa et al., 2015, 2016; Moneke et al., 2010). Some of the organochlorine pesticides known to be used for the pest management include dichloro-diphenyltrichloro-ethane (DDT), aldrin, dieldrin, endrin, heptachlor, hexachlorocyclohexane (HCH), toxaphene, endosulfan, and sodium pentachlorophenate. These pesticides can be degraded by different genera of bacteria (Table 19.2). Endosulfan and HCH are banned in many of the countries but in many regions are still in use (Castillo et al., 2011; Moneke et al., 2010; Kadam and Gangawane, 2005; Niewiadomska, 2004). Although wide-scale application of pesticides is an essential part of augmenting crop yield; an ideal pesticide should have the ability to destroy only target pest and should be able to undergo degradation to nontoxic substances as quickly as possible. Excessive use of these chemicals leads to the microbial imbalance, environmental pollution, and health hazards as well. Finally they enter the human and animal food chain causing neurotoxicological disorders. Farmers use chemical fertilizers to increase production, but the extensive use of these chemical-based inputs or fertilizers leads to contamination of soil and groundwater, depletion of soil fertility, greenhouse effect, damage to the ozone layer, acidification and pollution of water resources, destruction of beneficial microorganisms, acidification of soil, and health hazards (Martin et al., 2011). Pesticides with their inhibiting properties of pests and insects also possess potential toxic effects on health and environment. Generally most of the pesticides, insecticides, and herbicides may enter the food chain because they are sprayed or spread across entire agricultural fields. The exposure to pesticides through agriculture crops leads to a number of health effects, environmental hazards by disturbing soil and water ecosystems. Distribution of pesticides to air also leads to air pollution.
19.5 IMPACT OF PESTICIDES ON ENVIRONMENT The impact of pesticides in air by means of pesticide drift occurs when residues of pesticides gets suspended into air and are transmitted by wind to other areas potentially posing threat to surrounding environment. Physical parameters such as weather conditions, temperature, wind velocity, and relative humidity of the place at the time of pesticide applications contribute to its spread. Low relative humidity and high temperature of the place results in evaporation of pesticides in higher amount. Some pesticides which are applied for the fumigation of soils can synthesis volatile organic compounds which form a pollutant matter called tropospheric ozone by reacting with other chemicals. Droplets of liquid pesticides sprayed to fields may adhere to dust and gets transmitted as dust particles.
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19. AZOTOBACTER—A NATURAL RESOURCE
19.6 IMPACT OF PESTICIDES ON SOIL AND WATER Soil is an important and primary source for pesticide contamination. Extensive application of pesticides causes deleterious effects effect to soil microorganisms and other natural microflora of the soil ecosystem by altering normal metabolism of the microorganisms. The incidence of pesticides in water bodies such as lakes, canals, and rivers is reported to cause threat to water bodies. The cause of entry of pesticide to water includes drifting of pesticide when sprayed, percolation through soil, water runoff, accidental spilling, or by soil erosion. All these factors lead to suffocation of aquatic biota and zooplankton due to their toxicity.
19.7 IMPACT OF PESTICIDES ON HUMAN BEINGS Pesticides that are applied to the fields enter our body through inhalation of dust aerosols and vapor by inhaling or through oral exposure by consuming pesticide contaminated foods and water. The severity of pesticide on human beings depends on the toxicity and chemical nature and the long-term duration of the exposure to the pesticide. The severity may be acute or long-term effects. Acute effects are headaches, nausea, abdominal pain, vomiting and dizziness, respiratory tract infection, sore throat, allergies, weakness, and skin and eye problems. Long-term effects include neurological disorders, reproductive effects, birth defects, fetal death, and other fertility-related problems. Cancer-related problems such as lymphoma, brain, prostate, liver, blood, and skin are also reported. Pesticides are also referred as endocrine disruptors since the consumption of these chemicals leads to hormone imbalance in the body (Martin et al., 2011; Naik et al., 2007; Aleem et al., 2003).
19.8 EFFECT OF PESTICIDES ON NATURAL BIODIVERSITY Chemical compounds adversely affect the population of Azotobacter in soil, such as pesticides, insecticides, fungicides, herbicides, and nematicides, which are being used worldwide for the management of many agricultural crops. The population of Azotobacter was affected by several factors in soil, among them the excess use of pesticides and chemical fertilizer for the management of agriculture crops pests and diseases are important one. In the context of soil, pests are fungi, bacteria, insects, worms, and nematodes, etc. that can cause damage to field crops. Thus, in a broad sense pesticides are insecticides, fungicides, bactericides, herbicides, and nematicides that are used to control or inhibit plant diseases, control weeds, and insect pests (Naik et al., 2007; Martin et al., 2011; Mrkovacki et al., 2002; Castillo et al., 2011). The effect of pesticides on different natural ecosystem (air water and soil) is represented in Fig. 19.2.
19.9 EFFECT OF INSECTICIDES ON IAA PRODUCTION Effect of insecticide (chlorpyrifos and phorate) on IAA production by Azotobacter species was observed at various concentrations (1%–5%) as compared to the control. The highest IAA was produced by Azotobacter salinestris isolate supplemented with 1 mg of tryptophan at 1% chlorpyrifos (28 μg/mL), as compared to control (33 μg/mL). This result indicates that the 1% chlorpyrifos did not affect the bacterial growth and activity but reduced the IAA quantity. All the isolates produced IAA in the range of 29.4–33 μg/mL in control and the production of IAA varied from 15.3 to 28 μg/mL at 1%–5% chlorpyrifos. Significant differences were recorded among the isolates at 3% and 5% phorate and A. salinestris produced a maximum amount of IAA (22 μg/mL) at 5% phorate (Chennappa, 2016). The phorate concentration has reduced bacterium’s respiration rate by 25%–30% (Fig. 19.3).
19.10 EFFECT OF INSECTICIDE ON NITROGEN FIXATION The highest N2 fixation was recorded by A. salinestris supplemented with 1% chlorpyrifos (27.8 μg N mL 1 day 1) under in vitro condition. Among all the five isolates, A. salinestris and A. armeniacus isolates showed similar nitrogen fixation at 5% chlorpyrifos concentration but the addition of higher concentration has reduced the activity of the bacteria when compared to the control. Highest nitrogen fixation was observed with A. salinestris (33.2 μg N mL 1 day 1) isolate and all the five isolates fixed nitrogen in the range of 30.3–33.2 μg N mL 1 day 1 at 1% phorate concentration
19.10 EFFECT OF INSECTICIDE ON NITROGEN FIXATION
FIG. 19.2
273
Effects of pesticides on environment and biodiversity.
FIG. 19.3
Effects of insecticides on IAA production activity of Azotobacter species.
274
19. AZOTOBACTER—A NATURAL RESOURCE
FIG. 19.4
Effects of insecticides on nitrogen-fixation activity of Azotobacter species.
(Fig. 19.4). As in case of phorate treatment, no significant growth reduction was observed between 1% and 3% concentration and very slight nitrogen-fixation variation was observed at 5% concentration (Chennappa, 2016).
19.11 EFFECT OF INSECTICIDES ON GA PRODUCTION All the five Azotobacter isolates produced GA in the range of 7.5–14.50 μg, 25 mL 1 supplemented with 1%–5% chlorpyrifos concentration. A. salinestris isolate produced the highest amount of GA (9.20 μg, 25 mL 1) at 5% chlorpyrifos as compared to the other isolates. A. salinestris isolate produced a maximum quantity of GA (14.5 μg, 25 mL 1) at 1% chlorpyrifos concentration. In comparison between 1% and 5%, there was a variation in GA production. Among different concentrations of chlorpyrifos, 5% has reduced the GA production efficiency of Azotobacter as compared to the control (Chennappa, 2016). Above 1% concentration of chlorpyrifos reduced GA production ability of the Azotobacter and also bacterial growth by 20%–25%. Among all the isolates, A. vinelandii produced least amount (6.0 μg, 25 mL 1) of GA which was supplemented with 5% phorate and showed sensitive toward the GA production (Fig. 19.5).
FIG. 19.5 Effects of insecticides on GA production of Azotobacter species.
19.14 BIODEGRADATION OF INSECTICIDES BY AZOTOBACTER SPECIES
275
19.12 EFFECT OF INSECTICIDE ON PHOSPHATE SOLUBILIZATION The formation of halo zone around the colony indicates the phosphate-solubilizing activity of isolates. The diameter of the clear zone was in the range of 7.1–13.5 mm in 7–8 days up to 5% of chlorpyrifos under in vitro condition. The phosphate solubilization (PS) of Azotobacter found maximum at 1% chlorpyrifos concentration and the activity was reduced up to 35%–40% at 3%–5% concentration (Chennappa, 2016). The growth of the Azotobacter was slightly reduced by the addition of 3%–5% phorate to the culture media but not at 1% (Fig. 19.6). The effect of phorate and chlorpyrifos was clearly noticed on PS activity and A. vinelandii, A. armeniacus isolates showed lesser zone (7.1 and 7.5 mm) as compared to the control (13.5 and 13.0 mm).
19.13 BIODEGRADATION OF PESTICIDES Microorganism’s plays a major role in the degradation of pesticides. For degradation of these hazardous pesticide compounds a number of microorganisms were used and some of the soil-borne bacterial strains such as Arthrobacter spp., Burkholderia spp., Bacillus spp., Azotobacter spp., Flavobacteria spp., Pseudomonas spp., and Rhodococcus spp. are widely used in the degradation and bioremediation studies (Chennappa et al., 2015; Castillo et al., 2011). These bacterial genera possess enzymes and functional genes which are responsible for the degradation of such toxic pesticides (Table 19.3).
19.14 BIODEGRADATION OF INSECTICIDES BY AZOTOBACTER SPECIES The Azotobacter armeniacus, Azotobacter tropicalis, Azotobacter chroococcum, Azotobacter vinelandii, and A. salinestris were used for biodegradation of chlorpyrifos (1% concentration). Assay showed that all the Azotobacter strains degraded chlorpyrifos compounds efficiently. Among five isolates, A. tropicalis and A. salinestris showed highest biodegradation (95.2%–97.6%) of chlorpyrifos (Figs. 19.7 and 19.8). A. armeniacus showed low percent degradation (85.1%) of chlorpyrifos. The retention time of chlorpyrifos in gas chromatography was found to be 8.92–9.0 min in the sample peak area. None of the isolates showed 100% degradation of chlorpyrifos. However, A. armeniacus and A. salinestris showed 95%–97% biodegradation. Over all, all the isolates degraded chlorpyrifos in a range of 85%–97.6% and very minimum quantity of chlorpyrifos residues have been recovered from the treatment samples. In control no degradation of pesticides was observed (Chennappa, 2016; Chennappa et al., 2015, 2018b).
FIG. 19.6 Effects of insecticides on PS of Azotobacter species.
276 TABLE 19.3
19. AZOTOBACTER—A NATURAL RESOURCE
List of Bacterial Genera Encoded With Pesticide Degrading Enzyme
Genes
Bacterial genera
Encoded enzymes
References
Opd
P. diminuta
OPH
Serder et al. (1989)
Flavobacterium sp.
OPH
Mulbry et al. (1986)
F. blaustinum
OPH
Somara and Siddavattam (1995)
Pseudomonas sp.
OPH
Chaudry et al. (1988)
Alteromonas sp.
OPAA
Cheng et al. (1996)
A. haloplnaktis
OPAA
Cheng et al. (1997)
A. undina
OPAA
Cheng et al. (1996)
hocA
P. monteilli
ND
Horne et al. (2002)
Mpd
Plesiomonas sp.
ND
Zhongli et al. (2001)
adpB
Nocardia sp. B-1
ADase
Mulbry (1992)
PdeA
Delftia acidovorans
Phosphor diesterase
Tehara and Keasling (2003)
PepA
Escherichia coli
AMPP
Jao et al. (2004)
Phn
Escherichia coli
Phosphonatase
Chen et al. (1990)
Glp A
P. pseudomallei
C-P lyase
Penaloza et al. (1995)
pehA
Burkholderia caryophilli
PEH
Dotson et al. (1996)
opaA
FIG. 19.7
Biodegradation of chlorpyrifos by Azotobacter species.
100 97.6
98 96
95.2
Percent degradation
94 91.8
92 90
88
88 86
85.1
84 82 80 78 A. armeniacus
A. tropicalis
A. chroococcum Isolates
A. vinelandii
A. salinestris
277
REFERENCES
Chromatogram
Intensity 60,000
50,000
40,000
30,000
9.001 / Chlorpyriphos
20,000
10,000
0 0
FIG. 19.8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
min
GCMS analyses of chlorpyrifos degradation.
19.15 CONCLUSIONS Among the soil microflora PGPR possess a significant impact on physiological and structural properties of soil ecosystem. Due to its vast beneficial properties with the production of antifungal metabolites, amino acids, vitamins, IAA, nitrogen fixation, siderophores, and other growth-promoting substances with its ability to degrade the harmful pesticides it is considered as one of the alternative for the safe and sustainable agriculture practices. In this study the potential ability of Azotobacter species in degrading pesticides has been highlighted. Most of the Azotobacter species in the soil rhizosphere can break down toxic pesticides and substituted phenolic compounds into simpler inorganic microelements. However, the extensive use of such pesticides can create a greater threat to mankind and will certainly cause ecological imbalance in the nature. Therefore, this study reveals that the application of such Azotobacter spp. will certainly serve as a safe alternative in the management of diseases and with its potent plant growth properties.
References Agarwal, N., Singh, H.P., 2002. Antibiotic resistance and inhibitory effect of Azotobacter on soil borne plant pathogens. Indian J. Microb. 42, 245–246. Ahmad, F., Ahmad, I., Khan, M.S., 2005. Indole acetic acid production by the indigenous isolated of Azotobacter and fluorescent Pseudomonas in the presence and absence of tryptophan. Turkish J. Biol. 29, 29–34. Ahmad, F., Ahmad, I., Khan, M.S., 2008. Screening of free living rhizospheric bacteria for their multiple growth promoting activities. Microbiol. Res. 173–181. Aleem, A., Isar, J., Malik, A., 2003. Impact of long term application of industrial wastewater on the emergence of resistance traits of Azotobacter vinelandii isolated from rhizosphere soil. Bioresour. Technol. 86, 7–13. Almon, L., 1958. The vitamin B12 content of Azotobacter vinelandii. J. Nutri. 643–648. Anwar, S., Liquat, F., Khan, Q.M., Khalid, Z.M., Iqbal, S., 2009. Biodegradation of chloropyrifos and its hydrolysis product 3, 5, 6- trichloro-2pyridinol by bacillus pulmilus strain C2A1. J. Hazar. Mat. 168, 400–405. Bagyaraj, D.J., Patil, R.B., 1975. Azotobacter research in Karnataka. Curr. Res. 4, 181–184. Barazani, O., Friedman, J., 1999. IAA is the major root growth factor secreted from plant growth mediated bacteria. J. Chem. Ecol. 25, 2397–2407. Barrera, D.A., Soto, E., 2010. Biotechnological uses of Azotobacter vinelandii current state limits and prospects. African J. Biotechnol. 9, 5240–5250.
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Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorous nerve agents and nucleotide sequence of the enzyme. App. Env. Microb. 62, 1636–1641. Cheng, T.C., Rastogi, V.K., DeFrank, J.J., Anderson, D.M., Hamilton, A.B., 1997. Nucleotide sequence of a gene encoding and organophosphorous nerve agent degrading enzyme from Alteromonas haloplanktis. J. Indian Microb. Biotechnol. 18, 49–55. Chennappa, G., 2016. Impact of pesticides on Azotobacter species isolated from paddy field soil samples [Thesis]. University of Mysore, Mysore. Chennappa, G., Adkar-Purushothama, C.R., Naik, M.K., Sreenivasa, M.Y., 2014. Impact of pesticides on PGPR activity of Azotobacter Sp. isolated from pesticide flooded paddy soils. Greener J. Biol. Sci. 4 (4), 117–129. Chennappa, G., Adkar-Purushothama, C.R., Suraj, U., Tamilvendan, K., Sreenivasa, M.Y., 2013. Pesticide tolerant Azotobacter isolates from paddy growing areas of northern Karnataka. India. World. J. Microbiol. 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Microb. 42 (6), 574–576. Horne, I., Harcourt, R.L., Sutherland, T.D., Russell, R.J., Oakeshott, J.G., 2002. Isolation of a Pseudomonas monteilli strain with a novel phosphotriesterase. FEMS Microb. Lett. 206, 51–55. Jao, S.C., Huang, L.F., Tao, Y.S., Li, W.S., 2004. Hydrolysis of organophosphate triesters by Escherichia coli aminopeptidase P. J. Mol. Catabol. Enzymatic. 27, 7–12. Johri, B.N., Sharma, A., Virdi, J.S., 2003. Rhizobacterial diversity in India and its influence on soil and plant health. Adv. Biochem. Eng. Biotechnol. 84, 49–89. Kadam, T.A., Gangawane, L.V., 2005. Degradation of phorate by Azotobacter isolates. Indian J. Biotechnol. 4, 153–155. Khan, H.R., Mohiuddin, M., Rahman, M., 2008. Enumeration, isolation and identification of nitrogen-fixing bacterial strains at seedling stage in rhizosphere of rice grown in non-calcareous grey flood plain soil of Bangladesh. J. Faculty. Env. Sci. Technol. 13, 97–101. Kraepiel, A., Bellenger, J., Wichard, T., Morel, F., 2009. 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19 Azotobacter—A Natural Resource for Bioremediation of Toxic Pesticides in Soil Ecosystems G. Chennappa*, Nidoni Udaykumar*, M. Vidya†, H. Nagaraja‡, Y.S. Amaresh§, and M.Y. Sreenivasa‡ *Department of Processing and Food Engineering, College of Agricultural Engineering, Raichur, India † Centre for Nanotechnology, Department of Processing and Food Engineering, Raichur, India ‡ Department of Studies in Microbiology, University of Mysore, Mysore, India § Department of Plant Pathology, College of Agriculture, Raichur, India
O U T L I N E 19.1 Introduction
267
19.8 Effect of Pesticides on Natural Biodiversity
272
19.2 Benefits of PGPR
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19.9 Effect of Insecticides on IAA Production
272
19.3 Plant Growth-Promoting Substances 19.3.1 Vitamins 19.3.2 Amino Acids 19.3.3 Indole Acetic Acid 19.3.4 Gibberellic Acid 19.3.5 Phosphate Solubilization 19.3.6 Natural Biocontrolling Agents 19.3.7 Hydrogen Cyanide 19.3.8 Siderophores
269 269 269 269 269 269 270 270 270
19.10 Effect of Insecticide on Nitrogen Fixation
272
19.11 Effect of Insecticides on GA Production
274
19.12 Effect of Insecticide on Phosphate Solubilization
275
19.13 Biodegradation of Pesticides
275
19.4 Effect of Pesticides
270
19.15 Conclusions
277
19.5 Impact of Pesticides on Environment
271
References
277
19.6 Impact of Pesticides on Soil and Water
272
19.7 Impact of Pesticides on Human Beings
272
19.14 Biodegradation of Insecticides by Azotobacter Species 275
19.1 INTRODUCTION Plant protection has been an integral part of agricultural practices and average yield losses in India are estimated to be 10%–30% which is caused by insects, diseases, and weeds. Nearly a total of 43.5% pesticides are used to protect cotton and 38.6% for protection of rice crop (Kadam and Gangawane, 2005). Presently different types of formulated pesticides are used worldwide to control wide spread of diseases and to minimize the economic losses
New and Future Developments in Microbial Biotechnology and Bioengineering https://doi.org/10.1016/B978-0-444-64191-5.00019-5
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© 2019 Elsevier B.V. All rights reserved.
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19. AZOTOBACTER—A NATURAL RESOURCE
TABLE 19.1
Use of Different Types of Pesticides in Agriculture and Their Target of Diseases
Sl. no.
Pesticides
Chemical nature
Target hosts
1
Pendimethalin
Herbicides
Broad leaves, Grass
2
Phorate
Nematodes
Hershieminella oryzae
3
Glyphosate
Herbicides
Cyanodondoctylon
4
Simazine
Herbicides
Grass plants
5
Malathion
Insecticides
Leaf hoppers
6
Carbendazim
Fungicides
Pericularia oryzae
7
Toxaphene
Insecticide
Fruit borer
8
Carbofuran
Nematodes
Hershieminella oryzae
9
Monocrotophos
Insecticide
Brown plant hopper
10
Endosulfan
Insecticide
Fruit and leaf borer
11
Chloropyrifos
Insecticide
Yellow stem borer
12
Methyl parathion
Insecticide
Stem and leaf borer
(Chowdhary et al., 2018; Bharagava and Mishra, 2018). Unscientific and inappropriate use of these pesticides may diminish the population of beneficial microorganisms present in the soil. Among them, chemical pesticides and chemical fertilizer reaching the soil in significant quantities have direct effect on soil microbiological aspects, environmental pollution, and health hazards. Due to its excessive use and its distribution in the environment it pollutes soil, water bodies, and enters the food chain causing neurotoxicological disorders in animals, birds, and human beings (Martin et al., 2011). Pesticides cause everlasting changes in the soil microflora (Aleem et al., 2003), adverse effect on soil fertility and crop productivity, inhibition of nitrogen-fixing bacteria (Sachin, 2009), interference with ammonification, adverse effect on mycorrhizal symbiosis in plants and nodulation in legumes (Reinhardt et al., 2008). Pesticide concentration alters the soil ecosystem, soil microbe interaction, plant growth and soil structure, organic matter decomposition, biogeochemical cycling of elements. Several toxic and carcinogenic pesticides have been banned in many of the countries including India. Number of microbial species has the ability to break down toxic into simpler nontoxic compounds (Chowdhary et al., 2018; Bharagava and Mishra, 2018). To overcome pesticide effects, several biodegradation works have been carried out in order to minimize the pesticides residues in food and feed. For biodegradation of toxic pesticides, several types of soil-borne bacteria and fungi are widely used such as species of Arthrobacter, Burkholderia, Bacillus, Azotobacter, Flavobacteria, Trichoderma, Pseudomonas, and Rhodococcus (Chennappa et al., 2015; Castillo et al., 2011; Kadam and Gangawane, 2005; Moneke et al., 2010; Bagyaraj and Patil, 1975; Ramaswami et al., 1977). The application of different pesticides against the target host is presented in Table 19.1. Among the PGPR group, genus Azotobacter has the potentiality to produce different types of plant growthpromoting substances such as amino acids, plant growth hormones, antifungal antibiotics, and siderophore and fixes atmospheric-free nitrogen in soil (Chennappa et al., 2013, 2014, 2016; Myresiotis et al., 2012). Azotobacter species happens to be the most dominant species in the rhizosphere soil and can biodegrade different types of pesticide and substituted phenolic compounds used for the management of plant diseases (Li et al., 1991). Hence, degradation of such pollutant plays an important role in agricultural practice to reduce the load of pesticides from the soil and to serve safe food to the world and clean environment for future generation (Sujata and Bharagava, 2016; Kumari et al., 2016; Goutam et al., 2018).
19.2 BENEFITS OF PGPR Plant growth-promoting rhizobacteria (PGPR) are known to produce different types of secondary metabolites under congenial conditions. These metabolites include vitamins, amino acids, plant growth hormones, antifungal metabolites, hydrogen cyanide (HCN), and siderophores. These plant growth-promoting substances have direct influence on overall plant growth of several agricultural crops (Chennappa et al., 2018a; Myresiotis et al., 2012; Ahmad et al., 2005).
19.3 PLANT GROWTH-PROMOTING SUBSTANCES
269
19.3 PLANT GROWTH-PROMOTING SUBSTANCES 19.3.1 Vitamins PGPR group are one of the beneficial bacterial genera and Azotobacter species are one of the important bacterial group of PGPR’s known to produce different types of vitamins under favorable conditions. Azotobacter vinelandii (ATCC 12837) and Azotobacter chroococcum (CECT 4435) strains produced B-group vitamins which include niacin (Vit B3), pantothenic acid, riboflavin, and biotin. Vitamins are essential compounds for the physiological functions of the living beings which are produced by several groups of bacteria (Revillas et al., 2000). Riboflavin is a vitamin B2 required for a wide variety of cellular processes and it plays a key role in metabolism of fats, ketone bodies, carbohydrates, and proteins, respectively. Genetically engineered Bacillus subtilis and Corynebacterium ammoniagenes are used for mass production of riboflavin by which they overexpress genes of the enzymes involved in riboflavin biosynthesis (Almon, 1958; Revillas et al., 2000).
19.3.2 Amino Acids Amino acids are also important components for all living beings. Amino acids are produced by various processes and different types of microbes are also able to produce amino acids. Species of Azotobacter species produces different types of amino acids under diazotrophic conditions whereas growth media amended with glucose as sole carbon source (Lopez et al., 1981). Species of A. vinelandii and A. chroococcum are known to produce aspartic acid, serine, glutamic acid, glycine, histidine, threonine, arginine, alanine, proline, cysteine, tyrosine, valine, methionine, lysine, isoleucine, leucine, and phenylalanine (Revillas et al., 2000).
19.3.3 Indole Acetic Acid Indole acetic acid (IAA) is important plant auxin produced by different groups of soil-borne bacteria commonly living in plant rhizosphere (Barazani and Friedman, 1999). The IAA synthesizing ability has been detected in many rhizobacterial species as well as in pathogenic, symbiotic, and free-living bacterial species. Saline soil is a rich source of IAA-producing bacteria with 75% of the bacterial isolates is reported to be active in IAA production. Azotobacter species are found to produce IAA in the range of 2.09–33.28 μg/mL (Spaepen et al., 2007; Chennappa et al., 2016). Most commonly, IAA-producing PGPR strains are known to increase root length resulting in greater root surface area which enables plants to access more nutrients from soil. The IAA is responsible for the division, expansion, and differentiation of plant cells, tissues, and stimulates root elongation (Ahmad et al., 2008). Among PGPR species, Azospirillum, Aeromonas, Burkholderia, Azotobacter, Bacillus, Enterobacter, Pseudomonas, and Rhizobium are the most studied IAA-producing bacterial species which have been isolated from different rhizosphere soils (Reshma et al., 2018; Ahmad et al., 2008; Ghosh et al., 2010).
19.3.4 Gibberellic Acid Another important auxin, that is, gibberellins produced by several types of bacteria which include a wide range of chemicals that are produced naturally within plant rhizosphere. Gibberellic Acid (GA) produced by fungi called Gibberella fujikuroi in rice plant was first discovered and reported by Japanese scientist Eiichi Kurosawa. Gibberellins are important for seed germination, enzyme production that mobilizes growth of new cells. GA promotes flowering, cellular division and seed growth (Upadhyay et al., 2009).
19.3.5 Phosphate Solubilization Phosphobacteria play a significant role in the biotransformation of phosphate to soluble phosphorous form and are capable of hydrolyzing organic and inorganic phosphorus from insoluble compounds. Species of A. chroococcum, B. subtilis, B. cereus, B. megaterium, Arthrobacter ilicis, E. coli, P. aeruginosa, E. aerogenes, and Micrococcus luteus were identified as phosphate-solubilizing bacteria (Garg et al., 2001; Kumar et al., 2000). Chennappa (2016) reported the maximum level of phosphate-solubilizing Azotobacter species.
270
19. AZOTOBACTER—A NATURAL RESOURCE
FIG. 19.1 Various plant growth-promoting attribute of Azotobacter sp.
19.3.6 Natural Biocontrolling Agents The production of antibiotics is considered as one of the most studied biocontrol mechanisms for combating phytopathogens. Azotobacter produces antifungal antibiotic compounds (2, 3-dihydroxybenzoic acid, aminochelin, azotochelin, protochelin, and azotobactin) which inhibit the growth of plant pathogenic fungi, viz., Aspergillus, Fusarium, Curvularia, Alternaria, and Helminthosporium (Nagaraja et al., 2016; Khan et al., 2008; Mali and Bodhankar, 2009; Kraepiel et al., 2009). The 2, 4-DAPG is one of the most efficient antibiotics produced by Pseudomonas and it has a wide spectrum of antifungal, antibacterial, and anthelmintic properties (Naik et al., 2013). Azotobacter species act as biocontrol agents for many plant pathogens and Azotobacter armeniacus inhibited growth of Fusarium verticillioides (Mali and Bodhankar, 2009; Agarwal and Singh, 2002).
19.3.7 Hydrogen Cyanide Species of Azotobacter, Alcaligenes, Aeromonas, Bacillus, Pseudomonas, and Rhizobium capable of producing HCN a volatile, secondary metabolite that suppresses the growth of microorganisms and also influences the growth and development of plants (Ahmad et al., 2008; Chennappa et al., 2018a, b).
19.3.8 Siderophores Siderophores are produced and utilized by bacteria and fungi as iron (Fe)-chelating agents which are produced in response to iron deficiency which normally occurs in neutral to alkaline pH soils ( Johri et al., 2003). Azotobacter excretes siderophores under limited iron conditions and A. vinelandii produced five different siderophores, viz., 2, 3-dihydroxybenzoic acid, aminochelin, azotochelin, protochelin, and azotobactin. The main applications of siderophores act as drug delivery agents, antimicrobial agents, and soil remediation (Mollmann et al., 2009; Kraepiel et al., 2009; Barrera and Soto, 2010; Page and Von Tigerstrom, 1988). Azotobacter species also produces poly-bhydroxybutyrate (intracellular polyester), alginate, and catechol (extracellular polysaccharide) compounds under favorable environmental conditions (Barrera and Soto, 2010). PHB are used in large-scale production of biodegradable and biocompatible thermoplastic used as substitute for polyethylene and polypropylene. All the above beneficial aspects of Azotobacter species has been depicted in Fig. 19.1.
19.4 EFFECT OF PESTICIDES To control pests and diseases, farmers are applying excess quantity of pesticides and chemical fertilizers as compared to the recommended dosages. Several pesticides are regularly applied for the cultivation, viz., pendimethalin, phorate, glyphosate, simazine, malathion, carbofuran, monocrotophos, diazinon, fenthion, phosphamidon, methyl parathion,
271
19.5 IMPACT OF PESTICIDES ON ENVIRONMENT
TABLE 19.2 Degradation of Pesticides by Different Bacterial Species Sl. no.
Bacteria
Pesticides
References
1
Micrococcus
Chloropyrifos
Guha et al. (1997)
2
Flavobacterium sp.
Glyphosate
Singh and Walker (2006)
3
Enterobacter strain B-14
Chloropyrifos
Singh and Walker (2006)
4
Alcaligenes faecalis
Glyphosate
Singh and Walker (2006)
5
Rhodococcus erythropolis sp.
Carbendazim
Zhang et al. (2013)
6
Klebsiella sp.
Chloropyrifos
Ghanem et al. (2007)
7
Pseudomonas sp.
Monocrotophos
Singh and Walker (2006)
8
Bacillus pumillus strain C2A1
Chloropyrifos
Anwar et al. (2009)
9
Lactobacillus bulgaris
Chloropyrifos
George et al. (2014)
10
Serratia sp.
Chloropyrifos
Gangming et al. (2007)
11
Agrobacterium sp.
Chloropyrifos
George et al. (2014)
12
Azotobacter chroococcum
Endosulfan
Castillo et al. (2011)
13
Bacillus circulans
Pendimethalin
Megadi et al. (2010)
14
A. chroococcum
Phorate
Kadam and Gangawane (2005)
chlorpyrifos, carbendazim, mancozeb, and azinophos methyl, etc. (Chennappa et al., 2015, 2016; Moneke et al., 2010). Some of the organochlorine pesticides known to be used for the pest management include dichloro-diphenyltrichloro-ethane (DDT), aldrin, dieldrin, endrin, heptachlor, hexachlorocyclohexane (HCH), toxaphene, endosulfan, and sodium pentachlorophenate. These pesticides can be degraded by different genera of bacteria (Table 19.2). Endosulfan and HCH are banned in many of the countries but in many regions are still in use (Castillo et al., 2011; Moneke et al., 2010; Kadam and Gangawane, 2005; Niewiadomska, 2004). Although wide-scale application of pesticides is an essential part of augmenting crop yield; an ideal pesticide should have the ability to destroy only target pest and should be able to undergo degradation to nontoxic substances as quickly as possible. Excessive use of these chemicals leads to the microbial imbalance, environmental pollution, and health hazards as well. Finally they enter the human and animal food chain causing neurotoxicological disorders. Farmers use chemical fertilizers to increase production, but the extensive use of these chemical-based inputs or fertilizers leads to contamination of soil and groundwater, depletion of soil fertility, greenhouse effect, damage to the ozone layer, acidification and pollution of water resources, destruction of beneficial microorganisms, acidification of soil, and health hazards (Martin et al., 2011). Pesticides with their inhibiting properties of pests and insects also possess potential toxic effects on health and environment. Generally most of the pesticides, insecticides, and herbicides may enter the food chain because they are sprayed or spread across entire agricultural fields. The exposure to pesticides through agriculture crops leads to a number of health effects, environmental hazards by disturbing soil and water ecosystems. Distribution of pesticides to air also leads to air pollution.
19.5 IMPACT OF PESTICIDES ON ENVIRONMENT The impact of pesticides in air by means of pesticide drift occurs when residues of pesticides gets suspended into air and are transmitted by wind to other areas potentially posing threat to surrounding environment. Physical parameters such as weather conditions, temperature, wind velocity, and relative humidity of the place at the time of pesticide applications contribute to its spread. Low relative humidity and high temperature of the place results in evaporation of pesticides in higher amount. Some pesticides which are applied for the fumigation of soils can synthesis volatile organic compounds which form a pollutant matter called tropospheric ozone by reacting with other chemicals. Droplets of liquid pesticides sprayed to fields may adhere to dust and gets transmitted as dust particles.
272
19. AZOTOBACTER—A NATURAL RESOURCE
19.6 IMPACT OF PESTICIDES ON SOIL AND WATER Soil is an important and primary source for pesticide contamination. Extensive application of pesticides causes deleterious effects effect to soil microorganisms and other natural microflora of the soil ecosystem by altering normal metabolism of the microorganisms. The incidence of pesticides in water bodies such as lakes, canals, and rivers is reported to cause threat to water bodies. The cause of entry of pesticide to water includes drifting of pesticide when sprayed, percolation through soil, water runoff, accidental spilling, or by soil erosion. All these factors lead to suffocation of aquatic biota and zooplankton due to their toxicity.
19.7 IMPACT OF PESTICIDES ON HUMAN BEINGS Pesticides that are applied to the fields enter our body through inhalation of dust aerosols and vapor by inhaling or through oral exposure by consuming pesticide contaminated foods and water. The severity of pesticide on human beings depends on the toxicity and chemical nature and the long-term duration of the exposure to the pesticide. The severity may be acute or long-term effects. Acute effects are headaches, nausea, abdominal pain, vomiting and dizziness, respiratory tract infection, sore throat, allergies, weakness, and skin and eye problems. Long-term effects include neurological disorders, reproductive effects, birth defects, fetal death, and other fertility-related problems. Cancer-related problems such as lymphoma, brain, prostate, liver, blood, and skin are also reported. Pesticides are also referred as endocrine disruptors since the consumption of these chemicals leads to hormone imbalance in the body (Martin et al., 2011; Naik et al., 2007; Aleem et al., 2003).
19.8 EFFECT OF PESTICIDES ON NATURAL BIODIVERSITY Chemical compounds adversely affect the population of Azotobacter in soil, such as pesticides, insecticides, fungicides, herbicides, and nematicides, which are being used worldwide for the management of many agricultural crops. The population of Azotobacter was affected by several factors in soil, among them the excess use of pesticides and chemical fertilizer for the management of agriculture crops pests and diseases are important one. In the context of soil, pests are fungi, bacteria, insects, worms, and nematodes, etc. that can cause damage to field crops. Thus, in a broad sense pesticides are insecticides, fungicides, bactericides, herbicides, and nematicides that are used to control or inhibit plant diseases, control weeds, and insect pests (Naik et al., 2007; Martin et al., 2011; Mrkovacki et al., 2002; Castillo et al., 2011). The effect of pesticides on different natural ecosystem (air water and soil) is represented in Fig. 19.2.
19.9 EFFECT OF INSECTICIDES ON IAA PRODUCTION Effect of insecticide (chlorpyrifos and phorate) on IAA production by Azotobacter species was observed at various concentrations (1%–5%) as compared to the control. The highest IAA was produced by Azotobacter salinestris isolate supplemented with 1 mg of tryptophan at 1% chlorpyrifos (28 μg/mL), as compared to control (33 μg/mL). This result indicates that the 1% chlorpyrifos did not affect the bacterial growth and activity but reduced the IAA quantity. All the isolates produced IAA in the range of 29.4–33 μg/mL in control and the production of IAA varied from 15.3 to 28 μg/mL at 1%–5% chlorpyrifos. Significant differences were recorded among the isolates at 3% and 5% phorate and A. salinestris produced a maximum amount of IAA (22 μg/mL) at 5% phorate (Chennappa, 2016). The phorate concentration has reduced bacterium’s respiration rate by 25%–30% (Fig. 19.3).
19.10 EFFECT OF INSECTICIDE ON NITROGEN FIXATION The highest N2 fixation was recorded by A. salinestris supplemented with 1% chlorpyrifos (27.8 μg N mL 1 day 1) under in vitro condition. Among all the five isolates, A. salinestris and A. armeniacus isolates showed similar nitrogen fixation at 5% chlorpyrifos concentration but the addition of higher concentration has reduced the activity of the bacteria when compared to the control. Highest nitrogen fixation was observed with A. salinestris (33.2 μg N mL 1 day 1) isolate and all the five isolates fixed nitrogen in the range of 30.3–33.2 μg N mL 1 day 1 at 1% phorate concentration
19.10 EFFECT OF INSECTICIDE ON NITROGEN FIXATION
FIG. 19.2
273
Effects of pesticides on environment and biodiversity.
FIG. 19.3
Effects of insecticides on IAA production activity of Azotobacter species.
274
19. AZOTOBACTER—A NATURAL RESOURCE
FIG. 19.4
Effects of insecticides on nitrogen-fixation activity of Azotobacter species.
(Fig. 19.4). As in case of phorate treatment, no significant growth reduction was observed between 1% and 3% concentration and very slight nitrogen-fixation variation was observed at 5% concentration (Chennappa, 2016).
19.11 EFFECT OF INSECTICIDES ON GA PRODUCTION All the five Azotobacter isolates produced GA in the range of 7.5–14.50 μg, 25 mL 1 supplemented with 1%–5% chlorpyrifos concentration. A. salinestris isolate produced the highest amount of GA (9.20 μg, 25 mL 1) at 5% chlorpyrifos as compared to the other isolates. A. salinestris isolate produced a maximum quantity of GA (14.5 μg, 25 mL 1) at 1% chlorpyrifos concentration. In comparison between 1% and 5%, there was a variation in GA production. Among different concentrations of chlorpyrifos, 5% has reduced the GA production efficiency of Azotobacter as compared to the control (Chennappa, 2016). Above 1% concentration of chlorpyrifos reduced GA production ability of the Azotobacter and also bacterial growth by 20%–25%. Among all the isolates, A. vinelandii produced least amount (6.0 μg, 25 mL 1) of GA which was supplemented with 5% phorate and showed sensitive toward the GA production (Fig. 19.5).
FIG. 19.5 Effects of insecticides on GA production of Azotobacter species.
19.14 BIODEGRADATION OF INSECTICIDES BY AZOTOBACTER SPECIES
275
19.12 EFFECT OF INSECTICIDE ON PHOSPHATE SOLUBILIZATION The formation of halo zone around the colony indicates the phosphate-solubilizing activity of isolates. The diameter of the clear zone was in the range of 7.1–13.5 mm in 7–8 days up to 5% of chlorpyrifos under in vitro condition. The phosphate solubilization (PS) of Azotobacter found maximum at 1% chlorpyrifos concentration and the activity was reduced up to 35%–40% at 3%–5% concentration (Chennappa, 2016). The growth of the Azotobacter was slightly reduced by the addition of 3%–5% phorate to the culture media but not at 1% (Fig. 19.6). The effect of phorate and chlorpyrifos was clearly noticed on PS activity and A. vinelandii, A. armeniacus isolates showed lesser zone (7.1 and 7.5 mm) as compared to the control (13.5 and 13.0 mm).
19.13 BIODEGRADATION OF PESTICIDES Microorganism’s plays a major role in the degradation of pesticides. For degradation of these hazardous pesticide compounds a number of microorganisms were used and some of the soil-borne bacterial strains such as Arthrobacter spp., Burkholderia spp., Bacillus spp., Azotobacter spp., Flavobacteria spp., Pseudomonas spp., and Rhodococcus spp. are widely used in the degradation and bioremediation studies (Chennappa et al., 2015; Castillo et al., 2011). These bacterial genera possess enzymes and functional genes which are responsible for the degradation of such toxic pesticides (Table 19.3).
19.14 BIODEGRADATION OF INSECTICIDES BY AZOTOBACTER SPECIES The Azotobacter armeniacus, Azotobacter tropicalis, Azotobacter chroococcum, Azotobacter vinelandii, and A. salinestris were used for biodegradation of chlorpyrifos (1% concentration). Assay showed that all the Azotobacter strains degraded chlorpyrifos compounds efficiently. Among five isolates, A. tropicalis and A. salinestris showed highest biodegradation (95.2%–97.6%) of chlorpyrifos (Figs. 19.7 and 19.8). A. armeniacus showed low percent degradation (85.1%) of chlorpyrifos. The retention time of chlorpyrifos in gas chromatography was found to be 8.92–9.0 min in the sample peak area. None of the isolates showed 100% degradation of chlorpyrifos. However, A. armeniacus and A. salinestris showed 95%–97% biodegradation. Over all, all the isolates degraded chlorpyrifos in a range of 85%–97.6% and very minimum quantity of chlorpyrifos residues have been recovered from the treatment samples. In control no degradation of pesticides was observed (Chennappa, 2016; Chennappa et al., 2015, 2018b).
FIG. 19.6 Effects of insecticides on PS of Azotobacter species.
276 TABLE 19.3
19. AZOTOBACTER—A NATURAL RESOURCE
List of Bacterial Genera Encoded With Pesticide Degrading Enzyme
Genes
Bacterial genera
Encoded enzymes
References
Opd
P. diminuta
OPH
Serder et al. (1989)
Flavobacterium sp.
OPH
Mulbry et al. (1986)
F. blaustinum
OPH
Somara and Siddavattam (1995)
Pseudomonas sp.
OPH
Chaudry et al. (1988)
Alteromonas sp.
OPAA
Cheng et al. (1996)
A. haloplnaktis
OPAA
Cheng et al. (1997)
A. undina
OPAA
Cheng et al. (1996)
hocA
P. monteilli
ND
Horne et al. (2002)
Mpd
Plesiomonas sp.
ND
Zhongli et al. (2001)
adpB
Nocardia sp. B-1
ADase
Mulbry (1992)
PdeA
Delftia acidovorans
Phosphor diesterase
Tehara and Keasling (2003)
PepA
Escherichia coli
AMPP
Jao et al. (2004)
Phn
Escherichia coli
Phosphonatase
Chen et al. (1990)
Glp A
P. pseudomallei
C-P lyase
Penaloza et al. (1995)
pehA
Burkholderia caryophilli
PEH
Dotson et al. (1996)
opaA
FIG. 19.7
Biodegradation of chlorpyrifos by Azotobacter species.
100 97.6
98 96
95.2
Percent degradation
94 91.8
92 90
88
88 86
85.1
84 82 80 78 A. armeniacus
A. tropicalis
A. chroococcum Isolates
A. vinelandii
A. salinestris
277
REFERENCES
Chromatogram
Intensity 60,000
50,000
40,000
30,000
9.001 / Chlorpyriphos
20,000
10,000
0 0
FIG. 19.8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
min
GCMS analyses of chlorpyrifos degradation.
19.15 CONCLUSIONS Among the soil microflora PGPR possess a significant impact on physiological and structural properties of soil ecosystem. Due to its vast beneficial properties with the production of antifungal metabolites, amino acids, vitamins, IAA, nitrogen fixation, siderophores, and other growth-promoting substances with its ability to degrade the harmful pesticides it is considered as one of the alternative for the safe and sustainable agriculture practices. In this study the potential ability of Azotobacter species in degrading pesticides has been highlighted. Most of the Azotobacter species in the soil rhizosphere can break down toxic pesticides and substituted phenolic compounds into simpler inorganic microelements. However, the extensive use of such pesticides can create a greater threat to mankind and will certainly cause ecological imbalance in the nature. Therefore, this study reveals that the application of such Azotobacter spp. will certainly serve as a safe alternative in the management of diseases and with its potent plant growth properties.
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