Comparative Degradation of Endosulfan by Mutant Microorganism and their Parents

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

• Degradation in soil

  Results in Tables 4 and 5 show the half lives of Endosulfan and the counts of microorganism. The parent strains of both bacteria and fungi (from stock culture) were the most effective in reducing the half lives of both a and β-Endosulfan. The parent bacteria strains caused 83% reduction in half lives of Both a and β-Endosulfan while the parent fungi were capable of causing up to 69 and 78% reduction in half lies of a and β-Endosulfan respectively.

  The general count of parent isolates of bacteria and fungi were high (80.3 × 10⁴ for bacteria and 0.3 × 10⁴ for fungi) compared the general counts of the most tolerate strains (0.9 × 10⁴ for bacteria and 0.01 × 10⁴ for fungi).

  Generation of sulphate from degradation of Endosulfan was monitored for a total of 60 days. In the control sets sulphate starts appear before 10th day and gradually increases with time and did not appear to decrease until 60 days.

  The highest level of sulphate observed was 0.2 mMl/l. However incubating Endosulfan with various isolates of tolerant strains caused a clear change in the level and fate of sulphate. The level of sulphate recorded was 0.2 mM l/l observed after 60 days of incubation with fungi isolate from 600mg/l Endosulfan (Fig. 7)

• Degradation in carbon free media

  Results in Table 6 and 7 show the half lives of Endosulfan and the general counts of microorganism. The parent strains of both bacteria and fungi (from stock culture) were most effective in the reduction of half lives of both α and β-Endosulfan. The parent bacterial strains caused up to 61.2 and 83% reduction in half lives of both a and β-Endosulfan respectively. While the parent fungi were capable of causing up to 55 and 77%. reduction in half lives of α and β-Endosulfan respectively. The parent bacteria counts and fungi were high (63.2 × 10⁴ for bacteria and 0.1 × 10⁴ for fungi) compared to counts of the most tolerate strains (0.6 × 10⁴ for bacteria and 0.01 × 10⁴ for fungi).

  Generation of sulphate from degradation of Endosulfan was monitored for a total of 60 days. In control set sulphate starts to appear before 10^th day and gradually increases with time and did not appear to decrease until 60 days. The highest level of sulphate observed was 0.2 mM l/l.

  Tolerant strains caused a clear change in the level and fate of sulphate generated (Fig 9). The level of sulphate recorded was 0.09 mMl/l observed after 45 days of incubation with bacteria isolated from 600 mg/l eudosulfan (Fig 14)

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

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)

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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