Biological treatment of organ chlorinated pesticideUsing local bacterial isolates

Iman H. Qatia,Saad H. Khudair ,Nibal KH. Mosa and Ansam S. Sabie;Shahad Sh.Sabbar.

Ministry of science- Baghdad- IraqAbstract

The aim of this research was to isolate microbial isolates with ability of

growing in medium with presence of added pesticide and may therefore be used for

bioremiation of pesticides contaminated sites (Soil and water).

A bacterial groups which capable of degradation of chlorinated organic

pesticides was isolated from many agricultural soil and contaminated water.

After purification the strains were assessed in order to discover their ability to

degrade (COP) (α propachlor) with concentration of 25mg/L as carbon source in

mineral medium and in rich medium. However, the best three pure strains were able

to grow in M.M includes (COP) without enrichment.

This group which was composed of three isolates characterized based on

their morphological and biochemical characteristic. The isolates were presumptively

identified as rhodococcus spp. and Streptomyces albus. Using growth curve as a

parameter of (COP) compounds, optimum conditions (Pesticide concentration,

temperature, and time) of selected degrading bacterial strains were studied. Results

indicated that the optimal temperature was 37°C for all isolates, with best growth at

100mg/L of propachlor by str. albus, in addition of its efficiency to degrade the

compound during 48 hr at incubation.

Key words:biotreatment,bacteria,pesticides,soil pollutants

Introduction

The class of natural and synthetic chemical compound called pesticides holds

a very important place in agriculture and economics sati factory crop yields are

impossible without the use of pesticides despite the public an awareness that now

exists about harmful effects of pesticides use (Richins et al,1997). Many chlorinated

pesticides have been banded for use because of their short and long toxicity,

carcinogenicity, and environmental persistence. Despite the fact that most of these

chlorinated pesticides are now illegal to use, organ chlorines are still potential source

of pesticides poisoning (Munch and Engel, 1987 ,Surakha et al 2008).

The primary goal of our study was to develop an approach for enhancing

pesticide degradation using microorganisms. Isolation of indigenous bacteria capable

of metabolizing chlorinated organ pesticide has received considerable attention

because these bacteria provide an environmentally friendly method by detoxification.

Biodegradation is an economic friendly, cost effective, high efficient approach and

can be considered a superior alternative to physical and chemical methods which are

not only technically laborious and costly; also are not sufficient to completely degrade

organic toxins.

Complete biodegradation of pesticides involve the oxidation of parent compound

to form carbon dioxide and water. This process provides both carbon and energy of

the growth and reproduction of microbes if appropriate microorganism is absent in

soil or if biodegrading microbial population has been reduced due to toxicity of

pesticide in that case a specific microorganism can added or introduced in soil to

enhance the activity of the existing population ([Singh, 2008).The use of the bacteria

for the degradation and detoxification of numerous toxic chemical such as pesticides

is an effective tool to decontaminate the Polluted sites( Mervat , 2009). Degradation

by microbes depends not only on the presence of degradative enzymes, but also a

wide range of environmental parameters; temperature, pH, water potential, nutrients

and the amount of pesticide or metabolite in soil may also act as limiting factor for

pesticide degrading microorganisms, which requires further exploration and their

biochemical activities ( Singh 2008). A divers group of bacteria, including members

of the genera Alcaligenes, flavobacterium, Pseudomonas, and Rhodococcus,

metabolize pesticides. (Richins et al,1997, Malchandan and Kaneva , 1999).

Actinomycetes have considerable potential for the biotransformation and

biodegradation of pesticides (Richens et al,1997).

Fig (1) Schematic pathways proposed for the degradation of

propachlor. (Margarita et al 1999)

Co metabolism is the ability of microorganisms to catalyze transformation or

partial degradation of compound that do not support their growth. It is probably the

most wide spread mechanism for pesticide degradation (Horvath, 1971). Complete

mineralization of chemical is more likely to occur in mixed populations than with

single microorganisms (Adina and Rene 1999).

Materials and Methods

Soil sampling

4 soil samples were collected from potato and cornfield in some

Baghdad's farms, which had been treated with pesticides twice a year for

the previous 30 years and contaminated water from Tigris and Deala

River.

Preparation of

inoculums :

One gram of soil was suspended in 5ml of sterile (Mineral Medium) and this

suspension was considered the inoculums .The MM had the following composition

(per liter): 0.2g KH2PO4, 0.5g K2HPO4 (Sterilized separately at 125°C per 5 min. and

added to the rest of the salts; 1g (NH4)SO4;0.2g MgSO4.7H2O ;0.2g NaCl; 0.05g

CaCl2.2H2O; 0.025g FeSO4.H2O; 0.005g Na2MoO4; 0.0005g MnSO4(pH7±0.3).(Ortiz-

Hernandez et al,1993), were supplemented with 25mg/ L of (propachlor) as the

carbon source with or without Yeast extract (0.1%) in duplicate for each. ( Van –Elsas

and Smala (1997).

Water samples preparation :

One milliliter of water sample was added to 99 ml of D.W., mixed by vortexing and

regard as inoculums (Thomas,1996) .

Pesticide sampling:

One gram (gm) of Pesticide (propachlor) was weighted and filterized after added

to 25ml of methanol using (Millipore membrane, pore size 0.25mm) the methanol

was evaporated to dryness ( Ortiz-Hernandez and Sanchez-Salinas,2010).

Isolation of microorganisms:

Hundred milliliters of sabaroud broth for fungi isolation and similar flask contain L. Broth with 50 µg/L of antifungal (cycloheximide) for isolation of bacteria, supplemented with 25 mg/L of propachlor (in duplicate) and inculcate with 0.2 ml of each contaminated sample and incubated in shaker incubator at 37 ºc with 15 rpm for 7 days ,( Barnard B,2010).

Evaluation of Bacterial growth:

We carried out the growth and degradation experiment with the pure bacterial

colonies using flasks with 250 ml containing 100 ml of (MM) Mineral Medium

supplemented with series of pesticide dose and incubation at different temperature.

Cell growth was measured used spectrophotometer at O.D 600 and CFU/ml. After

preparing of serial fold dilution of liquid culture in D.W. cell growth of Streptomyces

was measured by CFU/ml only.

Characterization of isolates

The morphology of bacterial isolates was determined characterized by some

biochemical tests( Holt et al. 1994).

Results and discussion

Three soil samples, representing different agricultural soil and tow contaminated water samples, were using as inoculums to isolate microorganisms capable of utilization type of chlorinated organ pesticides( propachlor).

After culturing samples in special media. there is no any observed fungal

isolates. There was five bacterial isolates in addition of tow strains of Actionmyces

which were resistant at 25mg/L of pesticide).

. Table 1 reveals the bacterial efficiency growing on mm media with or without carbon source; and shows that three of the isolates Rh1spp, Rh2 spp and Strept1, shows good growth without addition of carbon source for enrichment. It can be observed that when the bacteria were cultured in presence of a rich medium, Including a carbon source (0.1%of yeast extract), capacity of degradation will be increase.

Table (1) Bacteria efficiency of growing on MM (with or without carbon source)

Bacteria Isolates MM. without carbon source MM with carbon source

Flav. 1 6×104 7×106

Flav . 2 2×104 2×106

Rho1 2×106 8×107

Rho2 2×106 5×107

Rho3 3×105 3×106

Spp1 5×106 8×107

Spp2 4×105 6×106

The physiological base for co- metabolism is not well known, but the most

accepted hypothesis is related to the specificity of enzymes( Malchandan and

Kaneva , 1999).

(Table 2) shows the various phenotypical characteristic of the selected isolates. They were presumptively identified as Rhodococcus spp. and Streptomyces albus.

Table (2) Characterization of Bacteria Isolates

Isolates Type of test Result

Streptomyces albus Chain of spores +

Gram stain G+veGrowth at 40º +White aerial mycelium +Starch utilizing +Formation of organic acids +

Rhodococcus 1&Rhodococcus 2

Gram stain G+ve

Colony texture Smooth , pigmented, orange color

Casein degradation +Ramose fermentation -Growth at crystal violate 0.0001gm/l +

These strains are interesting since it has rarely been isolated from clinical samples

and therefore its pathogencity is low. Optimum conditions of selected isolates were

studied, including (time, temperature and concentration of pesticide). Figure (2),

shows the efficiency of Streptomyces albus and Rho1, through their high growth

during first 48hr, in the presence of pesticide.

Fig. (3) shows results obtained from measuring growth rate of the bacterial culture, it can be seen that is the most growth in comparison to other strains on MM containing

100mg/L of pesticide, belong to Strept1 isolate. Strains showed the best growth at.

37ºC without significant differences between them fig.(4). It would be useful

to test these strains with other organic pesticides in order to find catalytic activity that

might make it recommendable treatment of wastes or polluted environment, with a

low potential effect public health. It has been observed that shortly after repeated

applications of degradable pesticide, the soil become richer in bacterial populations

which are capable of degradation. So it would dramatically reduce the effectiveness of

subsequent pesticide applications (Seth Nathan, 1989).

This study concludes that the isolated microbes can be used for biodegradation

and bioremediation of pesticides contaminated soil or water. The results also

suggested that is the co metabolism increase the ability of bacterial utilizing of

pesticide, so we can culture the strains as a consortium, several of bacteria

interfering processes, so there is no need to add nutrients.

Environmental conditions, soil pH, agricultural management of pesticides added

are important factor for bacterial use of xenobiotic compounds (such as

pesticides)as a growth substrate.

عزالت باستخدام المكلورة العضوية للمبيدات االحيائية المعالجة

بكتيرية

, , موسى خليل نبال خضير حسين سعد كاطع هندي صبحي ,ايمان انسام

صبار شكري وشهد سبع

- العراق بغداد والتكنولوجيا العلوم وزارة

الخالصــــــــة

وسط في النمو على القابلية لها محلية مجهرية أحياء عزل الى الحالي البحث يهدف

, تفكيك على قادرة العزالت هذه تكون وبهذا كربوني كمصدر المبيدات على يحوي غذائي

. على العثور تم مياه أو تربة كانت سواء بها الملوثة المناطق من المبيدات وأزالة

ملوثة و مختلفة ومياه تربة نماذج من عزلها تم التي البكترية العزالت من مجموعة

. نقيت, العزل أثناء الخمائر أو الفطريات من نوع اي على الحصول يتم لم حيث بالمبيدات

التجربة , هذه في المستخدم المبيد تفكيك على قابليتها ودرست البكترية العزالت هذه

وسط), propachlorوهو ( بأستخدام التجربة أعادة الى بأالضافة المعدنية األمالح وسط في

. النمو من فقط عزالت ثالث تمكنت اخر كربوني بمصدر وسط غني في جيدة بصورة

النمو من األخرى العزالت تتمكن لم و وحيد كربوني كمصدر المبيد على الحاوي األمالح

. بدراسة العزالت شخصت النمو يشجع آخر كربوني مصدر بوجود او ضعيفة االبصورة

للجنس , تعود منها اثنان ان ووجد البيوكيميائية الفحوصات وبعض المظهرية خواصها

Rhodococcus : , للنوع تعود فهي األخرى العزلة درست . Streptomyces aslbusاما

, ( ان ( , , لوحظ حيث الثالثة للعزالت المبيد تركيز الوقت الحرارة درجة المثلى الظروف

الحرارة للعزلتين ºم37درجة للعزلة افضل نمو مع الثالثة للعزالت المفضلة و Rho1هي

albus Streptomyces ال على , 48خالل االخيرة قابلية الى باالضافة للحضانة األولى ساعة

البالغ للمبيد التركيز عند اعلى بكفاءة . 100mg/lالنمو

, , , : والمياه التربة ملوثات المبيدات البكتريا االحيائية المعالجة المفتاحية الكلمات

References

-Adina, De. Schrijver and Rene De. Mot. (1999).Degradation of pesticides by Actionmycetes . Critical reviews in microbiology, 25(2); 85-119.

- Barnard B,2010.Isolation of Antibiotic-Producing Organisms from Soil. The

national health museum.

- Holt,J.G.,Krieg,N.R.Sneath,P.H,StaleyJ.T and Williams,S.T 1994.Bergeys

Manual of Determinative Bacterioogy.9thed.Baltimore.MD:Williams and

Wilkins.

- Horvath, R.S. 1971 Microbial contaminated of 2,4,5-6 trichlorophenoxy-

acetate acid , Bull. Environ. Contam. Toxical . , 5,537.

- Malchandan; A., Kaneva I., chen. W. (1999). Organophosphate pesticides by

immobilized Escherichia coli expressing organophosphate pesticides

hydrolyses on cell surface. Biotechnol. Bioeng. , 63; 261-223.

- Margarita M, Mengs G,  Allende J,  Fernandez J, Alonso R, and Ferrer E,

1999. Characterization of Two Novel Propachlor Degradation Pathways in

Two Species of Soil Bacteria. 65(2): 802–806.Appl Environ Microbiol.

- Mervat SM.2009 . Degradation of maltophilia M/ Elect. J. Biotech. , 12(4,1-

6).

- Munch D. J and T. Engel, (1987). (USEPA, Office of Water) (Battelle

Columbus Laboratories) –National Pesticide Survey Method 2, Revision 2.

- 0rtiz-Hernandez Ma .Luara and Sanchez-SalinaEnrique.2010.Biodegradation

of the organophosphate pesticide tetrachlorvinphos by bacteria isolated from

agricultural soils in Mexico.Rev.Int.Contam.Ambient.26(1) 27-38.

- Richins D., Kaneva I., Mulchandani A., and Chen W., (1997).Biodegradation

of organophsphprus pesticides by surface-expressed organ phosphorus

hydrolates .Nature biotechnol . 15, 984-987.

- Seth Nathan N. 1989.Biodegradation of pesticides in tropical Rice

Ecosystem (247-264).

- Singh D.K. 2008.Biodegradation and bioremediation of pesticides in soil.

Concept, method and recent development. Indian J. Microbiol. , 48; 35-40.

- Surakha R.M.,.akshm L, Surarnalath D. , Jaya M. , Aruna S. J, yothi K. ,

Narasimha G. and Venkates Warluk. 2008, Isolation and characterization of

chlorpyrifes degrading bacterium from agricultural soil and its growth

response. Afr. J., Microbiol. Res. 2, (26-31).

- Thomas G.W.1996 Soil PH and soil acidity. In Spark,D.L.(Ed),Methods of

soil analysis, Soil science society of America Book Series ,American Society

of agronomy and soil science society of America, Madison ,Wisconsin,5;475-

490

- Van –Elsas J.D. and Smala K. (1997) Methods for sampling soil microbes.

In: Manual of Environmental Microbiology ,(Chursty, G. Knudsen, M.

Mclnerney, L. Stetzenbach and M. Walter, Eds. J.American Society of

microbiology. Washington, D.C. pp.383-390. 

.