Comparative Degradation of Endosulfan by Mutant Microorganism and their Parents

By Dr. Osama El Gialani Elsaid¹, and Dr. Azhari Omer Abdelbagi²
November 2008

  1. Faculty of Agricultural Technology and Fish Sciences, Al-Neelain University, Khartoum, Sudan
  2. Faculty of Agriculture, University of Khartoum, Khartoum, Sudan
Abstract
Tolerant strains (mutant) of bacteria and fungi from the soil of Rass Elfeel pesticide store (Sudan, Mangil scheme) were isolated through consecutive exposure to elevated concentration of Endosulfan under condition of carbon free media and the results showed that the most tolerant fungi (can tolerate up to 1000 mg/l) was Aspergillus fumigates while the most tolerant bacteria was Bacillus sp. The comparative degradation of Endosulfan by tolerant strains and their parents was studied under condition of soil and carbon free media. Results showed that parent strains (present in large number) showed faster decrease in half lives compared to tolerant strains (few numbers). However tolerant strains might have greater potential if they find a chance to propagate in massive numbers.

Introduction

The chlorinated cyclic sulfite diester Endosulfan is a cyclodien insecticide possessing a relatively board spectrum of activity. Technical-grade is a mixture of two stereo isomers, α and β-Endosulfan, in a ratio of 7:3. It is used extensively throughout the world as a contact and stomach insecticide and an acaricide on field crops, vegetables, and fruit crops. Because of its abundant usage and potential transport, Endosulfan contamination is frequently found in the environment at considerable distances from the point of its original applications (Mansingh and Wilson, 1995; Miles and Pfeuffer, 1997). Endosulfan has been detected in the atmosphere, soils, sediments, surface and rain water, and food stuffs. Its is extremely toxic to fish and aquatic invertebrates (Sunderam et al., 1992) and has been implicated in mammalian gonadal toxicity (Sinha et al., 1997), genotoxicity (Chaudhuri etal., 1999), and neurotoxicity (Pual and Balasubramaniam, 1997). These health and environmental concerns have led to an interest in detoxification of Endosulfan in the environment.

The biodegradation of persistent compounds is an important mechanism for their dissemination in the environment (Alexander, 1981; Marcae, 1990; Wallnofer& Engelhardt, 1990). In predicting the persistence of synthetic chemicals in soil, sediment and natural water, it is necessary to determine the role of endogenous microorganisms in the over all degradation process.

Microorganisms play an important role in the conversion of cyclodiene insecticides in soil to nontoxic products. In the natural environment microorganisms may provide some protection against toxicity of Endosulfan. Pure culture of a range of soil microorganisms have been reported to transform Endosulfan to a nontoxic diol metabolite in unsealed liquid cultures (Elzorgani & Omer, 1974 and Marten, 1976). Endosulfan can be completely degraded in about two weeks to nontoxic metabolite under anaerobic conditions (Guerin & Kennedy, 1999). Microbial degradation of Endosulfan was also reported by Shivaramaiah and Kennedy (2006). They ,also, identified endodiol as the major degradation product in an undefined mixture of microorganisms obtained from soil suspension. Tariq et al. (2000) reported that degradation of Endosulfan occurred in contaminant with bacterial growth when Endosulfan was used as only source of sulfur in the culture, while no growth occurred in the absence of Endosulfan.
(Martens,1976) investigated the ability of 28 soil fungi, 14 soil bacteria, and 10 soil actinomycetes to degrade insecticide Endosulfan. He found that the major metabolites detected were Endosulfan sulfate.

Material and Methods

Mutant strains procedure

From stock cultures (isolated microorganism form soil by selective media) one ml was taken and placed in sets of sterilized test tubes containing CHB (Chabecks) media for fungi and MPB (Meat Peptone Agar) media for bacteria. The media were prior treated with 200 mg/l Endosulfan. The test tubes were incubated for seven days at 30° C. The growth of microorganisms in these test tubes was observed by locking for turbidity of the media. Then counts of microorganisms in each test tube were estimated. One ml was taken from each of the seven days incubated test tube and transferred to sterilized set of another test tubes each containing ten milliliter of CHB media (for fungi) or MPB (for bacteria) treated with higher concentration of Endosulfan (400 mg/l). Test tubes were incubated for another seven days, growth and count of microorganisms were observed and recorded.

Microorganisms were subject to further consecutive elevated concentrations of Endosulfan (600, 800 or 1000 mg/l) and effects on growth and counts were determined. Units were arranged in a completely randomized design with three replicates.

Identification of microorganisms

The microorganism tolerant to high concentrations of the Endosulfan were identified as follows.

  1. Bacteria

    Culture of sample on nutrient agar media
    One ml was taken by sterilized pipette from tests tubes containing the most tolerant microorganisms (test tube containing 1000 mg/l Endosulfan) and placed in a Petri dish containing sterilized nutrient agar. The inoculated plates were then incubated at 37° C for 24 hours. This procedure was replicated four times. The plates were checked for shape, colour and other general characteristics of the colonies growth.

    Gram stain
    The colonies obtained in the four plates were then subjected to Gram stain test as described in Brough (1999). One drop of distilled water was added to sterilized slides, and then small portion of colony was taken by the loop on a drop of water, and then was spreaded over the slide. The drop was allowed to dry by exposure to air at room temperature. Then the smear was fixed by heating on a flame and stained by three types of stains (Crystal violet stain for 60 seconds, lugols iodine for 60 seconds, and decolorized by alcohol for 10 seconds). After each stain the smear was rapidly washed by water. Lastly the smear was dried by exposure to air and examined under oil by microscope 100 × magnifications. The slides were examined for Gram positive rod with central and terminal to sub terminal spores and results were recorded.

    Inoculation in Mannitol salt agar
    Small portion of colonies grown in nutrient agar plates were taken by sterilized loop and inoculated in Mannitol salt agar. They were incubated at 37° C over night. Shape and colour of colonies were recorded.

  2. Fungi

    Culture in PDA
    One ml was taken from test tubes containing the most tolerant fungi (test tube containing 1000 mg/l Endosulfan), and placed in a Petri dish containing sterilized PDA media. The inoculated plates were incubated at 25° C for seven days. This procedure was repeated four times. The plates were checked daily for hyphal shape and colour.

    Lacto phenol cotton blue stain (LPCB)
    One drop of LPCB was placed in a sterilized slide, then small amount from the growing culture were taken using a loop and placed in the LPCB drop. The Slide was then covered and examined under microscope at 10 × and 40 × magnifications for hyphal characteristic.

The comparative degradation experiment

The purpose of this experiment was to study the relative capability of tolerant stains (compared to their parents) degrading Endosulfan in the liquid media and soil.

  1. Degradation under liquid media

    A pre cleaned and sterilized conical flask (500 ml) was prepared. Three hundreds ml liquid media (carbon free media) was placed in the flask. The flask with its contents was autoclaved at 121° C for 20 minutes. Then allowed to cool at room temperature. Five milliliter acetone containing 150 mg Endosulfan were added to sterilized media, and then acetone was evaporated by gentile flame. The treated media was Sub-divided into 30 sets each composed of 10 ml in sterilized test tubes. Test tubes in triplacate were then treated with Endosulfan (500 mg/l) and inoculated with one ml of either bacteria or fungi as follows;

    1. Bacteria

      1. Organic nitrogen bacteria (parents)
      2. Organic nitrogen bacteria exposed to 200 mg/l Endosulfan
      3. Organic nitrogen bacteria exposed to 600 mg/l Endosulfan
      4. Organic nitrogen bacteria exposed 1000 mg/l Endosulfan
        (tolerant strains)
    2. Fungi

      1. Fungi (parents)
      2. Fungi exposed to 200 mg/l Endosulfan
      3. Fungi exposed to 600 mg/l Endosulfan
      4. Fungi exposed to 1000 mg/l Endosulfan
  2. Degradation under soil conditions

    A pre cleaned and sterilized conical flask (1000 ml) containing 500 g soil was prepared. The flask with its contents was sterilized in an oven at 160° C for three hours. The flask was allowed to cool at room temperature. Two hundreds ml distilled water containing 150 mg Endosulfan were added to sterilized soil. The treated soil was sub-divided into 30 sets each composed of 10 g in sterilized flasks (50 ml). Flasks in triplicate were then treated with Endosulfan (500 mg/kg) and inoculated with one ml of either bacteria or fungi as follows:

    1. Bacteria

      1. Organic nitrogen bacteria (parents).
      2. Organic nitrogen bacteria exposed to 200 mg/l Endosulfan
      3. Organic nitrogen bacteria exposed to 600 mg/l Endosulfan
      4. Organic nitrogen bacteria exposed to 1000 mg/l Endosulfan
    2. Fungi

      1. Fungi (parents).
      2. Fungi exposed to 200 mg/l Endosulfan
      3. Fungi exposed to 600 mg/l Endosulfan
      4. Fungi exposed to 1000 mg/l Endosulfan

All test tubes and flasks were arranged in a completely randomized design with three replicates and incubated at 30° C for a total of 60 days. The level of starting material and Endosulfan sulphate generated was checked at 15 days interval.

Extraction and analysis
All flasks were incubated at 30° C for 60 days and residues of Endosulfan were extracted and analyzed using GLC every 15 days.

Results

Identification of tolerant strains capable of growing at elevated level of Endosulfan
Fungal and organic nitrogen bacteria from stock culture of selected soil types (Ras Alfeel pesticide store) were exposed to elevated concentration (200, 400, 600, 1000 mg/l) of Endosulfan in carbon free media. The general growth, counts and shape of colonies observed.

Identification of organic nitrogen bacteria tolerate to high concentration of Endosulfan
The organic nitrogen bacteria were cultured according to the methods described before (Brough, 1999).
Results of various steps of culturing leading to the identification as Bacillus sp was summarized in table 1.

Identification of tolerant fungi
The fungi tolerant to elevated level of Endosulfan was identified following the methods of Brough.(1999). Summary of the test done and main observation leading to the identification of the fungus as Aspergillums fumigatus in table 2.
Less tolerates fungal, types; Mocursp and Aspergillus niger were tentatively identified based on the color of the hyphae (white hyphae for Mocur sp. and Black hyphae for Aspnegillus niger).

Counts of identified tolerant microorganism
Results of counts of identifies tolerant types are summarized in table 3.
It is clear that the various types of fungi have different tolerance to elevated level of Endosulfan; Mocur sp. can tolerate up to 400 mg/l, Aspregillus niger can tolerate up to 600 mg/l Aspergillums fumigatus can tolerate up to 1000 mg/l. Counts generally decrease with increasing the concentration of Endosulfan in the media.
However the only bacteria tolerant to the highest level of Endosulfan (1000 mg/l) was Bacillus sp.
Other unidentified types of bacteria were observed at lower concentration. Bacterial counts also seen to decrease when the concentration of Endosulfan in the media increased.

Comparative degradation of Endosulfan by tolerant strains and their parents

Table 1 · Identification tests for tolerant organic nitrogen bacteria
Test sequence Culture test Observations
Ist Test Culture in nutrient agar media Dry, white and creamy colonies
2nd Test Gram stain Gram positive rod with central and terminal to sub terminal spores.
3rd Test Inoculation in Manito salt agar Yellow, flat dry colonies
Table 2 · Identification test for tolerant fungi and main observation
Test sequence Test done Observations
Ist Test Inoculation in PDA In face green- yellowing hyphen
2nd Test Lacto phenol cotton blue stain Septet. Hyphen. Phased containing conidia spores
Table 3 · General counts of Fungi & Organic Nitrogen bacteria tolerant to the highest concentration of Endosulfan (1000 mg/l)
Types of microorganisms Concentration (mg/l)
Zero 200 400 600 800 1000
Fungi Mocur + + + + + +
Aspregillus Niger + + + + + + + + +
Aspregillus fugamugatus + + + + + + + + + + + + + +
Bacteria Bacillus Sp. + + + + + + + + + + + + + +
+ = Growth covering less than 25 % of plate
++ = Growth covering between 25 – 50 % of the plate
+++ = Growth covering above 50 % of the plate
– = no growth observed
Table 4 · Half live (days) and percentage reduction in half live of α-Endosulfan incubated with microorganisms isolated from elevated concentration of Endosulfan in soil
Microorganisms Slope τ ½ (days) Reduction in τ ½ % No. of Cells per gm soil
Organic Nitrogen Bacteria from stock culture 0.7173 1.5 14.4 82.7 80.3 × 104
Organic Nitrogen Bacteria isolated from 200 mg/l Endosulfan 0.8947 0.99 39.7 52.2 4.9 × 105
Organic Nitrogen Bacteria isolated from 600 mg/l Endosulfan 0.8097 1.2 28.7 65.4 6.7 × 104
Organic Nitrogen Bacteria isolated from 1000 mg/lEndosulfan 0.8862 1.4 33.3 59.9 0.9 × 104
Fungi from stock culture 0.9800 1.6 26.0 68.7 0.3 × 104
Fungi isolated from 200 mg/l Endosulfan 0.9687 1.5 29.6 64.4 0.1 × 104
Fungi isolated from 600 mg/lEndosulfan 0.7671 1.6 16.6 79.9 0.1 × 104
Fungi isolated from 1000 mg/l Endosulfan 0.8775 1.2 30.9 62.7 0.1 × 104
Controls 0.9529 0.54 82.9 0.0 0.0
R² = Determination coefficient
τ ½ = Half lives
Table 5 · Half live (days) and percentage reduction in half live of β-Endosulfan incubated with microorganisms isolated from elevated concentration of Endosulfan in soil
Microorganisms Slope τ ½ (days) Reduction in τ ½ % No. of Cells per gm soil
Organic Nitrogen Bacteria from stock culture 0.7753 1.54 15.4 82.5 8.0 × 105
Organic Nitrogen Bacteria isolated from 200 mg/l Endosulfan 0.8331 0.89 35.5 59.6 4.9 × 105
Organic Nitrogen Bacteria isolated from 600 mg/l Endosulfan 0.8754 1.54 18.1 78.9 6.7 × 104
Organic Nitrogen Bacteria isolated from 1000 mg/L Endosulfan 0.9737 1.69 30.4 65.4 0.9 × 104
Fungi from stock culture 0.8910 1.61 19.4 77.9 0.3 × 104
Fungi isolated from 200 mg/l Endosulfan 0.7900 1.74 20.6 76.6 0.1 × 104
Fungi isolated from 600 mg/lEndosulfan 0.9223 1.59 22.2 74.8 0.1 × 104
Fungi isolated from 1000 mg/l Endosulfan 0.8403 1.82 30.7 65.1 0.01 × 104
Controls 0.9921 0.53 87.9 0.0 0.0
R² = Determination coefficient
τ ½ = Half lives
Table 6 · Half live (days) and percentage reduction in half live of α-Endosulfan incubated with microorganisms isolated from elevated concentration of Endosulfan in Carbon-free media
Microorganisms Slope τ ½ (days) Reduction in τ ½ % No. of Cells per gm soil
Organic Nitrogen Bacteria from stock culture 0.7535 0.91 35.4 61.2 6.3 × 105
Organic Nitrogen Bacteria isolated from 200 mg/l Endosulfan 0.7026 0.81 39.9 56.2 3.5 × 105
Organic Nitrogen Bacteria isolated from 600 mg/lEndosulfan 0.8909 0.76 63.2 30.6 4.9 × 104
Organic Nitrogen Bacteria isolated from 1000 mg/l Endosulfan 0.9333 1.1 41.6 54.3 0.6 × 104
Fungi from stock culture 0.9402 1.1 41.0 54.9 0.1 × 104
Fungi isolated from 200 mg/l Endosulfan 0.7014 0.93 31.9 64.9 0.4 × 103
Fungi isolated from 600 mg/l Endosulfan 0.9673 1.1 47.8 47.5 0.4 × 103
Fungi isolated from 1000 mg/l Endosulfan 0.9231 0.68 62.9 27.4 0.1 × 103
Controls 0.9266 0.49 91.1 0.0 0.0
R² = Determination coefficient
τ ½ = Half lives
Table 7 · Half live (days) and percentage reduction in half live of β-Endosulfan incubated with microorganisms isolated from elevated concentration of Endosulfan in Carbon-free media
Microorganisms Slope τ ½ (days) Reduction in τ ½ % No. of Cells per gm soil
Organic Nitrogen Bacteria from stock culture 0.7698 1.6 16.8 82.5 6.3 × 105
Organic Nitrogen Bacteria isolated from 200 mg/l Endosulfan 0.8862 1.6 21.1 77.9 3.5 × 105
Organic Nitrogen Bacteria isolated from 600 mg/l Endosulfan 0.8346 1.4 37.5 60.8 4.9 × 104
Organic Nitrogen Bacteria isolated from 1000 mg/l Endosulfan 0.8137 1.6 17.6 81.7 0.6 × 104
Fungi from stock culture 0.9323 1.8 25.9 73.0 0.1 × 104
Fungi isolated from 200 mg/l Endosulfan 0.7883 1.4 21.7 77.3 0.4 × 103
Fungi isolated from 600 mg/l Endosulfan 0.9245 1.5 33.2 65.4 0.4 × 103
Fungi isolated from 1000 mg/l Endosulfan 0.8499 0.72 55.9 41.7 0.1 × 103
Controls 0.9936 0.50 95.8 0.0 0.0
R² = Determination coefficient
τ ½ = Half lives

Fig 1 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of organic nitrogen bacteria (Isolated from stock culture free from Endosulfan) in soil
Fig 1

Fig 2 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of organic nitrogen bacteria (exposed to 200 mg/l of Endosulfan) in soil
Fig 2

Fig 3 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of organic nitrogen bacteria (exposed to 600 mg/l of Endosulfan) in soil
Fig 3

Fig 4 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of organic nitrogen bacteria (exposed to 1000 mg/l of Endosulfan) in soil
Fig 4

Fig 5 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of fungi (Isolated from stock culture free from Endosulfan) in soil
Fig 5

Fig 6 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of fungi (exposed to 200 mg/l of Endosulfan) in soil
Fig 6

Fig 7 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of fungi (exposed to 600 mg/l of Endosulfan) in soil
Fig 7

Fig 8 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of fungi (exposed to 1000 mg/l of Endosulfan) in soil
Fig 8

Fig 9 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of organic nitrogen bacteria (Isolated from stock culture free from Endosulfan) in carbon-free media
Fig 9

Fig 10 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of organic nitrogen bacteria (exposed to 200 mg/l of Endosulfan) in Carbon-free media
Fig 10

Fig 11 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of organic nitrogen bacteria (exposed to 600 mg/l of Endosulfan) in Carbon-free media
Fig 11

Fig 12 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of organic nitrogen bacteria (exposed to 1000 mg/l of Endosulfan) in carbon-free media
Fig 12

Fig 13 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of fungi (Isolated from stock culture free from Endosulfan) in carbon-free media
Fig 13

Fig 14 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of fungi (exposedto 200 mg/L of Endosulfan) in carbon-free media
Fig 14

Fig 15 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of fungi (exposed to 600 mg/l of Endosulfan) in carbon-free media
Fig 15

Fig 16 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) with parent strain of fungi (exposed to 1000 mg/l of Endosulfan) in carbon-free media
Fig 16

Fig 17 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) in sterilized soil (control)
Fig 17

Fig 18 · Degradation of Endosulfan (α, β) and generation of sulphate upon incubation of the chemical (100 mg/l) in sterilized carbon-free media (control)
Fig 18

Discussion

Tolerant strains of bacteria and fungi from the soil of Ras Elfeel pesticide store were isolated through consecutive exposure to elevated concentration of Endosulfan. Following the criteria listed in Brough (1999) the most tolerant fungi (can tolerate up to 1000 mg/l) was identified as Aspergillus fumigatus, while the most tolerant Bacteria was identified as Bacillus sp. Other fungi tolerant to lower concentration of Endosulfan were tentatively identified as Asperegillus niger (600 mg/l) and Mocur sp. (up to 400 mg/l). The tentative identification was based on partial fulfillment of the criteria listed by Brough (1999).

The comparative degradation of Endosulfan by tolerant strains and their parents was studied in both soil and carbon free media. Results indicated that parents strains (present in more number) caused faster decrease in half lives compared to tolerant strains (found in lower number). Although the most tolerant isolates appear relatively less efficient, (compared to parent) but relating the counts with the capability in reducing half lives it appeared that they can be of a great potential if they had a chance to propagate in massive numbers. This explanation could be supported by the work of Tariq et al. (2000) who indicated that increasing the number of microorganism caused better activity and more degradation.

The sulphate was generated more under soil conditions compared that of carbon free media. Various factors may affected the generation of sulphate under soil conditions specially the rate of oxygen diffusion. Such condition may not be available in carbon free media.

The Sudanese isolates of microorganism could be of great potential in reducing the level of Endosulfan in highly polluted storage soils. The use of microorganism for bioremediation requires better and more understanding of all the physiological and biochemical aspects involved in chemical transformations. This work is attempt to put a corner stone for some aspects needed for bioremediation of polluted sites. However further studies are needed prior to start any bioremediation process in such sites.

Acknowledgement

Thanks are extended to those who contributed in one way or another to this work. They include Prof. Osman Ibrahim Gameel, Prof. Gafar Zorgani, Dr. Awad Galal, Dr. Imad Ali, Ismael Siddeg, Tarig Elsir and Triza.
My strong appreciation is extended to Lift Engineering Company for financing this work

References

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