Isolation, Degradation Characteristics And Immobilization Of Chlorpyrifos Degrading Bacteria

Organophosphorus pesticides are widely used in the world, due to their high efficiency and cheapness. However, the damage to the health of the person and the pollution to the environment can not be ignored. At present, several organophosphorus compounds have been successfully removed from soil and aquatic environments using degrading microbes. In recent years, lots of organophosphorus pesticides degrading bacterium have been isolated, such as Pseudomonas sp., Flavobacterium sp., Alcaligenes sp., Arthrobacter sp., Enterobacter asburiae, Sphingomonas sp., Rhodocouus rhodochrous and other strains. Nevertheless, because of the low fitness to the environment, the isolated strains can not meet the requirement of the pollution control. Therefore, it is urgent to get lots of microorganisms to degrading the pesticides. In addition, the literatures about the biodegradation of the Bacillus cereus strains are scarce, especially for the same genus but not the same strains. The aim of this study is to isolate the degrading bacteria of the organophosphorus pesticides, study the taxonomy of the strains, the effects of the environmental factors to the biodegradation, the degradation kinetics of the pesticide by degrading microbes and the construction of the mixed strain. In addition, the optimized culture medium and fermentation conditions for enzyme production and the enzyme characteristics are also studied. Finally, the immobilization of the degrading bacteria and its characteristics are investigated. The results are as follows:1. Three bacteria designated as HY-1, HY-2 and HY-4, respectively, were isolated from the wastewater treatment system of the pesticide enterprises. The traditional morphology, physiological-biochemical properties as well as 16S rDNA gene sequence analysis were applied to the bacteria classification, the characteristics of degrading methyl-parathion, chlorpyrifos and triazophos were also studied. The three strains were identified as the different strains of Bacillus cereus and the Genbank registration numbers were eu915687, eu915686 and eu915688, respectively. The degradation rates of HY-1, HY-2 and HY-4 reached 91.7%, 88.7% and 92.4% within 72 h when the medium contained 50 mg/L of methyl-parathion, the degradation rates among three strains had no significant difference at the 0.05 level. When the concentration of chlorpyrifos were 100 mg/L, the degradation rates of HY-1, HY-2 and HY-4 reached 64.8%,53.7% and 59.5% within 72 h, respectively. The degradation rates were from 13.3% to 20.7% within 72 h when the medium contained 100 mg/L of triazophos. The difference in the degradation rates of organophosphorus pesticides showed the diversity of the degradation characteristics of different Bacillus cereus strains.2. The effects of the environmental factors to the degradation rates of organophosphorus pesticides were studied with single factor test. The strain HY-2 could utilize methyl-parathion and chlorpyrifos as the unique phosphorus source. The appropriate conditions for strain HY-2 degrading methyl-parathion were as follows: the temperature 30-35°C, the initial pH value 6-8, the initial methyl-parathion concentration 10-50 mg/L and the inoculation amount was 20% of the total volume. Extra adding glucose could not enhance the degradation of methyl-parathion. The optimal conditions for strain HY-2 degrading chlorpyrifos were as follows: glucose concentration 6 g/L, the temperature 30-35°C, the initial pH value 7, the initial chlorpyrifos concentration 80-200 mg/L and the inoculation amount was 20% of the total volume. The optimal conditions for strain HY-1 and HY-4 degrading chlorpyrifos were as follows: glucose concentration 3 g/L, the initial pH value 7 to 8 , the temperature 35°C, the initial chlorpyrifos concentration 80 mg/L and 20% inoculation amount.The optimum content of the yeast extract was 1g/L when the concentration of the glucose was 3 g/L and the optimal concentration of the yeast extract was 5 g/L when the medium did not contain glucose for the strain HY-4. In the tested concentrations, the degradation rates to chlorpyrifos without glucose did not reach the level that containing glucose.3. The growth pattern of the bacteria and degradation kinetics of chlorpyrifos by the strain HY-1 and HY-2 were approached in the paper. Simultaneously, the tolerance to high concentrations of chlorpyrifos and the forming ability of zoogloea were also studied.The optimal incubation time of the seed culture of the three strains was 10 h, 19 h and 15 h, respectively. The inoculation amount of the prepared inocula was 8% of the total volume. The results showed that the adaptation period of the bacteria was prolonged by addition of chlorpyrifos. The logarithmic phase and stationary phase were also extended against the control. Moreover, the pH of the culture medium increased with the increase of the cell density. The degradation curve was different when the medium contained different concentrations of chlorpyrifos.The two bacteria had a sorption and desorption course during the degradation, especially at the high concentrations. When the initial concentrations of chlorpyrifos were 40, 80, 100 and 120 mg/L, the calculated results indicated that the first-order model gave a good fit. The strain HY-2 had a higher tolerable chlorpyrifos concentration, while the forming ability of zoogloea of strain HY-2 was lower than that of HY-1. The results suggested that the two microbes merited further study as the potential biological agents for the remediation of soil, water, or crop contaminated with chlorpyrifos.4. In order to clarify the degradation effect of the mixed culture of Bacillus cereus strains, the mixed culture of HY-1 and HY-4 was constructed by comparing the degradation rate of different ratio (V/V) of the two strains and the mixed culture of HY-1, HY-2 and HY-4 was also constructed by the orthogonal test. Furthermore, the effects of the inoculum size, pH value, initial chlorpyrifos concentration and salt content on the degradation efficiency were estimated by the single factor test. The results of the mixed culture of HY-1 and HY-4 were as follows: the optimized ratio (V/V) of HY-1 and HY-4 was 1:1. Addition of glucose stimulated the growth of the mixed strains but did not enhance the degradation efficiency of chlorpyrifos. The alkaline environment was prone to form the biomass and to achieve a high degradation rate of chlorpyrifos. The results indicated that the degradation characteristics of the mixed strain of HY-1 and HY-4 were similar to the characteristics of strain HY-1. When the sodium chloride concentration was ranged from 20 g/L to 70 g/L, the degradation rates of the mixed strain to 80 mg/L of chlorpyrifos were above 41%. The results of the mixed culture of HY-1, HY-2 and HY-4 were as follows: the optimal ratio (V/V) of the inoculum of the three strains was 1:1:3, inoculum size 8% (V/V), initial pH 7. The biodegradation process conformed to the first-order kinetic model in the tested concentrations. The degradation rates to 80 mg/L of chlorpyrifos were still high and could reach the highest up to 61% while the sodium chloride concentration of the culture medium increased from 20 g/L to 100 g/L. The results suggested that the mixed strain could be used as a potential and efficient chlorpyrifos degrader for the bioremediation of contaminated sites, especially for the cleanup of high salinity wastewater in the chlorpyrifos production enterprises.5. The optimal fermentation medium and fermentation conditions of HY-1 were selected by single factor and orthogonal test, and the amount of enzyme produced by HY-1 was estimated based on the specific activity of the enzyme. The optimal composition of the culture medium included glucose 6.0 g/L, tryptone 2.2 g/L, K2HPO4 2.0 g/L, KH2PO4 0.2 g/L, MgSO4·7H2O 0.1 g/L, NaCl 0.1 g/L and trace elements solution 2 mL/L. The optimal fermentation conditions of HY-1 were as follows: incubation time of the seed culture 16 h, fermentation time 18 h, inoculum size 1% (V/V), initial pH 7. The specific activity of the degrading enzyme was not affected while the sodium chloride concentration of the fermentation medium increased from 0 g/L to 30 g/L. To the best of our knowledge, this is the most extremely salt-tolerant bacterium reported that could degrade chlorpyrifos.6. In order to clarify the characteristics of the crude enzyme, the crude enzyme was extracted from the strain HY-1. The results indicated that the soluble protein content of the crude enzyme was determined with Albumin (bovine serum) as standard protein and the soluble protein of the crude enzyme was 2.21 g/L. The Km value and the maximal enzymatic degradation rate for chlorpyrifos were 1.2356 mmol/L and 0.0226μmol/(mg·min), respectively. The appropriate incubation time for the enzymatic degradation was 1 h. The highest specific activity of the crude enzyme was gained when the adding volume of the crude enzyme was 1 mL. The crude enzyme activity had optimal temperature of 28°C for the enzymatic degradation of chlorpyrifos. The degradation rate was still over 78% of the maximal activity within temperature ranged from 20°C to 44°C. The crude enzyme showed comparatively greatest enzymatic activity at pH 6, high activity when pH ranged from 5 to 9. Furthermore, additional experimental evidence revealed that the crude enzyme had some extent stability for temperature and pH. The crude enzyme also degraded chlorpyrifos efficiently while it was dealt with sodium chloride from 10 g/L to 70 g/L for 1 h.7. The Bacillus cereus HY-1 was immobilized with sodium alginate and the biodegradation of chlorpyrifos by Bacillus cereus immobilized with sodium alginate was investigated. The appropriate reaction time was obtained firstly. Meanwhile, the effects of various parameters, such as the amount of immobilized biomass, pH and the concentration of chlorpyrifos on the biodegradation were studied. The results showed that chlorpyrifos can be availably degraded by Bacillus cereus immobilized with sodium alginate. The time for chlorpyrifos to reach a steady concentration was 60 h. The degradation efficiency of chlorpyrifos was the highest when the amount of immobilized biomass was 160 g/L. The immobilized strain had a broader pH adaptation range. Compared with the high initial chlorpyrifos concentrations, the degradation rates of low initial concentrations (80 mg/L and 100 mg/L) of chlorpyrifos were higher. When the immobilized bacteria were repeated used for 4 times, the degradation rate to 100 mg/L chlorpyrifos was 47%. Compared with activated sludge, the method of using immobilized Bacillus cereus in sodium alginate produced less sludge and separated microorganism from water more effectively. Therefore, the immobilized strain had a great application value in the aspect of removing chlorpyrifos residue.