Friday, May 8, 2015

Endosulfan Degradation by Anaerobic Bacteria-Best Methodology



















By: Gomal Amin 

What is Endosulfan?
Endosulfan is a neurotoxic organochlorine insecticide, which is used worldwide for pest control and its residues have been remained for long periods in soil, water bodies and agricultural products. Endosulfan is highly insoluble in water. Hence, mostly it will be associated with soil. It is reported that, the half-life of soil bound Endosulfan was much higher than that of aqueous forms. Health and environmental concerns have led to an interest in detoxification of endosulfan in the environment.
Effect on Human Body:
High doses of endosulfan in humans primarily affect the central nervous system, resulting in respiratory depression, dry mouth, convulsions, and possibly death. Low doses have been shown to compromise the immune system, liver, kidneys and reproductive system in animals.
How it enters to Environment:
Endosulfan was used as potential pesticide, after its application on plants it will attach with soil particles and soil erosion, run-off, spray drift, and atmospheric deposition contribute to releases of endosulfan to aquatic ecosystems.
Degradation products: 
Endosulfan diol, Endosulfan sulfate, Endosulfan ether, and Endosulfan lactone

How Experiment is conducted and its conditions:
·        The extent of biodegradation of endosulfan was 80% in aqueous and 60% in soil slurry during 60 days in anaerobic conditions.
·        Pure as well as mixed microbial cultures utilized ES as a carbon and sulfur source in both aqueous and soil mediums.
·        Methanogens were identified in an anaerobic mixed culture degrading ES and the major degradation product was endosulfan diol (ESD) along with traces of, endosulfan sulfate, endosulfan ether, and endosulfan lactone
·        Study was done to obtain the kinetics of complete biodegradation pathways of ES in the aerobic and anaerobic environments, and identify the rate limiting steps so that the accumulations and persistence of the toxic metabolites could also be predicted.
·        The 100 g L-1 of anaerobic seed soils were used to inoculate the enrichment cultures in 200mL autoclaved basal nutrient media NS2
·        The composition of NS2 (Basal nutrient media) used to enrich anaerobic culture was (in mg L-1): KH2PO4 8300, K2HPO4 6800, NH4Cl 1300, MgSO4.7H2O 100, Fe2 (SO4)3 10, Na2 EDTA 15, Cu (NO3)2 1, ZnCl2 2.5, MnCl2 1, CoCl2 0.05, H3BO3 1, and Na2SO4 4. The pH of the solutions was adjusted to 7.
·        NS2was purged with nitrogen earlier to use. ES at a concentration of 0.491 mM was prepared using market grade ES, was added as a carbon source in anaerobic cultures. The culture was maintained at room temperature (~25 C’). The regular media replenishment was done at an interval ~15 d for anaerobic culture. After 6–8 cycles, the cultures were separated from media using streak plate on NS2 base containing 1.5% Agar. Then the colonies were moved to anaerobic environment.
·        After centrifugation and suspending in phosphorus buffer saline of neutral ph, the inoculums of bacterial culture is proliferated in different plates containing Anaerobic Agar.
·        The degradation products were extracted from solution using Soxhlet Extraction procedure (USEPA Method 3540).
·        Biodegradation experiments in aqueous cultures:
The anaerobic biodegradation experiments were conducted for 60d, respectively, in 40 mL borosilicate glass vials, crimp sealed with Teflon lined septa. In the aqueous phase experiments, each autoclaved vial contained 35 mL of 2.46l M of ES solution prepared in NS2 (anaerobic) media, and 1 mL of either microbial seed in PBS or autoclaved phosphorus buffer saline. The vials were placed on a rotary shaker at 20 rpm and kept at room temperature (~25 C’). At pre-determined sampling times, vials in duplicate were removed from the shaker, and samples were processed for extraction and analysis in GC–MS.

Pathway of degradation:
Hydrolysis to ESD was the only route of ES degradation in anaerobic medium which was achieved using first order kinetics model. The degradation pathway anaerobic degradation of ES was based on the time of appearance, peak position and centre of mass of concentration profiles of the metabolites.
In aqueous media, hydrolysis to the less toxic endosulfan diol is expected to be the dominant transformation pathway. For their potential degradation of endosulfan and its metabolites, i.e., endosulfan sulfate, endosulfan ether, and endosulfan lactone, in anaerobic condition.
Under anaerobic conditions, ES was hydrolyzed to ESD which was converted to ESE. ESS and ESL were not detected in the anaerobic system but according to some other experimental results the ESS presence is due to lack of oxygen or anaerobic condition.
A new unknown metabolite is also detected which is degradation product of ESE and/or ESL and is now known as ESU1.

Mass balance:
The carbon, chlorine and sulfur balance computed from the concentrations of the parent compound and identified metabolites showed that sulfur started to decrease in anaerobic aqueous cultures without any lag. The release of sulfur show that it will serve as food source for bacterial growth. On the other hand, no immediate loss of carbon and chlorine were recorded in any medium and the total carbon and chlorine started to decrease only after 15 d in anaerobic medium.
In the degradation pathways of ES to ESE in the anaerobic environment, no carbon and chlorine is lost. Loss of carbon and chlorine can only be attributed to further degradation of ESL or ESE to smaller products. These are also the times when significant build-up of ESU1 started in these systems, indicating that ESU1 was a degradation product of ESE/ESL.
Results:
Anaerobic mixed culture was able to degrade ~82% of the total ES in 60 d
References:
Paper name:    Kinetics of the biodegradation pathway of endosulfan in the aerobic
                        and anaerobic environments
Writers name: Manoj K. Tiwari, Saumyen Guha
Journal name: Elsevier –Chemosphere
Publication: Chemosphere 93 (2013) page No 567–573





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