A Feasible Way of Degrading Malathion Pesticide under Laboratory Condition Using Phosphate Solubilizing Bacteria

An experiment was carried out for identification and determination of malathion degrading phosphate solubilizing bacteria isolated from the agricultural fields. In this study, malathion degrading phosphate solubilizing bacteria were identified using NBRIP (National Botanical Research Institute’s phosphate growth medium) media. A number of bacterial colonies were screened from agricultural fields. From primarily screened colonies 4 isolates were identified as phosphate solubilizing bacteria through qualitative and quantitative analysis. The isolated 4 bacterial colonies were inoculated in NBRIP broth media enriched with malathion pesticides to observe degradation of malathion pesticide under incubation study at three different temperatures (25 ̊C, 30 ̊C and 37 ̊C). However, all the four isolates showed capability in degrading malathion pesticide. The study clearly revealed that phosphate solubilizing bacteria can be used in bioremediation of environmental pollution caused by malathion pesticide.


Introduction
Bangladesh is facing many challenges in agricultural sector in the coming years.
In order to secure food demand for its vast population, more toxic chemicals are used that have reputations of speed and effectiveness to increase food production.The Government of Bangladesh also promotes the use of pesticides to increase output per acre of land.Overall, pesticide use in general is increasing.They provide unquestionable benefit for agricultural production, even though, as a consequence, low amounts of some residues may persist in the food supply, air, water and soil and could constitute a significant exposure pathway for humans (Ghorab & Khalil, 2016).The growing use of pesticides in contemporary agriculture has direct or indirect impact on the composition of soil microorganisms.It is stated that usually more than 10 9 microorganisms are present per gram of soil representing 4000 to 7000 different genomes and biomass of 300 to 3000 kg per ha (Ranjard & Richaume, 2001) which directly or indirectly are affected by many conditions of these insecticides used.If pesticides can be degraded as soon as they are applied, then their desired function will not be served.
Pesticide biodegradation by microorganisms has been documented in a wide range of habitats, including soils, ground water and sewage sludges etc. Insecticides and their degradation products generally get accumulated in the top soil and influence not only the population of various groups of soil microbes but also their biochemical activities like nitrification, ammonification, decomposition of organic matter and nitrogen fixation (Agnihotri et al., 1981).Recently, use of microbes for effective detoxifying, degrading and removal of toxic compounds from contaminated soil and water has emerged as an efficient technique to clean up polluted environments (Strong & Burgess, 2008).
one of the common pesticides used in pest control programs in Bangladesh.In this study malathion was degraded by using Phosphate solubilizing bacteria under laboratory condition.As it is reported that phosphate solubilizing bacteria are capable of mineralizing organic phosphorous compound, so it is expected that these bacteria will be able to degrade organophosphate malathion releasing soluble phosphorous.
So, the efficiency of the isolated microorganisms to metabolize malathion in order to develop safe, convenient and economically feasible methods for pesticide detoxification would be investigated under this study.

Methods and Materials
Soils were collected from the area of Sirajganj, Tangail and Madhupur (Figure 1).All samples were collected from agricultural fields, where the common agricultural crop is paddy rice (Oryza sativa); other minor crops were also present in some of the fields such as Lady's finger (Hibiscus esculentus), Amaranthus (Amaranth sp) or Indian spinach (Basellaceae sp).

Malathion Pesticide
Malathion (57%) a commercial grade pesticide used mainly for pest control was collected from local pesticide shop from Dhaka, Bangladesh.The pesticide belongs to organophosphate class of pesticides.

Characterization and Identification of Phosphate Solubilizing Bacteria
The samples were collected from 8 -10 cm depth using a sterile spatula and transferred to autoclaved Ziplock bag stored under room temperature.Figure 3 illustrates the flowchart of soil collection and processing for identification of phosphate solubilizing bacteria.Soil samples were air dried and crushed before sieving.0.5 mm sieve was used for microbiological analysis and 2 mm sieve was used for determination of chemical properties of soil.To isolate bacteria, serial dilution plate technique was carried out (Greenberg et al. 1980).When colonies appeared, they were counted and recorded and were purified through repeated plating (by streak plate method) (Cappuccino & Natalie, 2012).These isolated colonies were identified by some morphological and biochemical tests such as shape, arrangement, colonies, form, margin, elevation, colour, Gram reaction, Furthermore, quantitative and qualitative tests were carried out by using National Botanical Research Institute's phosphate growth medium (NBRIP) to identify phosphate solubilizing bacteria.By measuring the amount of phosphorous solubilized and appearance of halo zones, the identified bacteria were considered as phosphate solubilizing bacteria (Figure 4).
Inorganic phosphate in the culture supernatant was estimated following the vanadomolybdophosphoric yellow color method using a spectrophotometer as described by Jackson (Jackson, 1958).The amount of inorganic phosphate solubilized in the supernatant was measured from the difference of sample and control media.The increase in inorganic phosphate in the supernatant was taken as the efficiency of microorganism in degrading malathion pesticide.Journal of Geoscience and Environment Protection

Isolation and Identification of Phosphate Solubilizing Bacteria
All soil conditions may not be suitable for growing all types of microorganisms.Vikram et al. (2007) reported a strong positive correlation between organic carbon content, available N and PSB population.Also, Ndung'u-Magiroi et al.
(2012) showed a positive and highly significant correlation between PSB and phosphate-solubilizing microorganism (PSM) populations, organic C, exchangeable Ca and exchangeable Mg, while pH and extractable P did not correlate with the PSB population.Winogradsky detected saprophytic Bacillus species in soil, stating that the bacteria's spores became active when an excess of easily decomposable organic matter became available or when the moisture content of the soil was high (Winogradsky, 1924).Pseudomonas aeruginosa is found widely in nature, in soil and water and has few nutritional requirements and can adapt to conditions not tolerated by other organisms (Prince, 2012).Therefore, the chemical parameters of soil samples were measured to determine favorable condition for the phosphate solubilizing Bactteria (Table 1).Among the 5 isolates, only 1 heterotrophic isolate was Gram-positive and rest were Gram-negative aerobic bacteria.Two bacteria named as Raoultella ornithinolytica and Pseudomonas pseudoalcaligenes isolated from agricultural soil of Sirajgonj, Pseudomonas pseudoalcaligenes and Bacillus cereus were isolated from moist soils of Tan- gail and lastly Pseudomonas aeruginosa and Bacillus subtilis were isolated from Journal of Geoscience and Environment Protection relatively moist agricultural field of Gazipur.Next, phosphate solubilizing bacteria were identified from isolated bacteria.Table 2 shows that out of the 5 isolates 4 showed phosphate solubilizing capacity.These were Raoultella ornithinolytica, Bacillus cereus, Pseudomonas aeruginosa and Bacillus subtilis.Bacillus cereus was the most efficient phosphate solubilizing bacteria (Figure 6).

Observation of Bacterial Response to Malathion Degradation
In the present experiment, malathion was the sole phosphorous source for microbes in the broth.The amount of soluble phosphate released in the medium proved the degradation of malathion.Malathion degradation was observed at 25˚C, 30˚C and 37˚C which were presented in Tables 3-5.The phosphorous releasing capacity of these microbes varied with timings and incubation temperature.
Figure 6.Organic phosphorous solubilizing capacity of Phosphate solubilizing bacteria.At 25˚C and 30˚C, the amount of soluble phosphate in the medium increased on the fourth day of incubation and then decreased gradually.This indicated that organisms showed their highest degradation activity on the fourth day of incubation when incubation temperature was fixed at 25˚C and 30˚C [Figure Temperature is one of the major environmental factors that influence biodegradation.Furthermore, bacterial metabolism may increase with an increase in temperature (Nadalian, et al., 2016).In the experiment, temperature was found to be a significant factor.It is reported that Bacillus species mainly grow in an optimum temperature of nearly 25˚C to 30˚C (Web, 1), Pseudomonas aeruginosa at 25˚C to 37˚C (Prince, 2012) and Raoutella ornithinolytica at 37˚C (Chung, et al., 2016).However, by measuring the amount of soluble phosphorous throughout the incubation period, it was demonstrated that for all organisms, 25˚C and 30˚C were the most favorable temperatures for degrading

Conclusion
It is reported that hydrolysis plays an important role in malathion degradation when pH > 7.This is because malathion is responsive to hydrolysis reaction because of its stronger polar nature.Although malathion may be degraded by chemical processes in soil such as chemical hydrolysis, the amount of microbial degradation is far greater than chemical degradation in natural systems (Mulla, et al., 1981).Biodegradation also plays a role when pH < 7 and the rate of hydrolysis is slow, relative to the rate of biodegradation (Web, 2).Different types of enzymes have been reported in organic phosphorous compound degradation.
These enzymes can either be acid or alkaline phosphomonoesterases (Jorquera, et al., 2011).Under this study efficiency of phosphate solubilizing bacteria in degrading malathion has been investigated.Release of inorganic phosphorous im-P.Chakma et al.

Sirajganj:
Soil sample was collected from agricultural field of Sirajganj.The GPS location of the sampling point where soil samples were collected was approximately 24˚24'27.7164''Nand 89˚38'7.1376''E.Sirajganj falls under Precambrian platform and is characterized by sedimentary rocks above a Precambrian igneous and metamorphic basement.The area lies in the active delta region of Journal of Geoscience and Environment Protection

Figure 1 .
Figure 1.Location of the sampling site.

Figure 3 .
Figure 3.A Flowchart of soil collection and processing for identification of phosphate solubilizing bacteria.

Figure 4 .
Figure 4. Presence of phosphate solubilizing bacteria in NBRIP solid media.

Figure 5 .
Figure 5. Malathion rich broth culture filtration for determination of soluble phosphate.

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(a) and Figure 7(b)].In contrast, at 37˚C all organisms showed the highest degradation capacity on the second day of incubation except Bacillus Subtilis which degraded most of the pesticide on the sixth day [Figure 7(c)].

Figure 7 .
Figure 7. Amount of phosphate released by phosphate solubilizing bacteria at 25˚C (a); 30˚C (b) and 37˚C (c).
malathion [Figures 8(a)-(d)].The release of the maximum amount of soluble Journal of Geoscience and Environment Protection

Table 1 .
Chemical properties of collected soil samples.

Table 2 .
Qualitative and quantitative screening of phosphate solubilizing bacteria.

Table 3 .
Release of soluble phosphorous by bacterial isolates at 25˚C.

Table 4 .
Release of soluble phosphorous by bacterial isolates at 30˚C.

Table 5 .
Release of soluble phosphorous by bacterial isolates at 37˚C.