Microbial Degradation Of Typical Chlorinated Hydrocarbons Pollutants In Second Songhua River

Chlorinated hydrocarbons in Second Songhua River are of diverse structure and multiple kinds. Most of them are characterized by high toxicity, reluctant degradation, and easy bioaccumulation and can do damage to other living things including mankind through bio-magnification. To remedy environmental pollution by this kind of pollutants in order to decrease risk and to prevent them from going into environment with man-made wastes are the main tasks for polluted water bodies control and prevention. Microorganisms play an important role in degradation and transformation of these matters existing in environment and wastes. This research isolated bacterial strains capable of degrade chlorinated hydrocarbons by using selective enrichment technology, and identified these strains by means of molecular biology methods, and studied the degradation characterization of strains in conditions of pure culture, gravel contact oxidation method simulation system and microcosm simulation system. The major conclusions are as following:(1) Isolated three bacterial strains with the capability of degrading chlorobenzene, nitrobenzene, and hexachlorobutadiene, respectively. By observing colony characterizations, determining partial physiological and biochemical indicators, and determining sequence of 16S rDNA and construct phylogenetic tree, three strains were identified as Acinetobacter calcoaceticus CB001, Pseudomonas putida NB001, and Serratia marcescens HL1.(2) The ranges of pH value for strains CB001 and HL1 to satisfactorily degrade chlorobenzene and hexachlorobutadiene, and for strain NB001 to degrade nitrobenzene were from 7.0 to 8.0 and from 7.0 to 7.5, respectively. The satisfactory range of temperature for three strains was from 25.0℃to 30.0℃, but strain CB001 grew well even at the temperature of 40.0℃. By acclimation at low temperature strains could satisfactorily degrade substrates even at temperature of 10.0℃and 15.0℃. Acclimation in culture medium containing chlorinated hydrocarbons played an important role in the biodegradation of substrates.(3) Growth of strain cells could be characterized by Lag phase, accelerated phase, exponential phase, and stationary phase and degree of change of substrates concentration correspondingly included slowness phase, rapidness phase, slowness phase. The maximal rate of biodegradation for strains CB001 and NB001 to degrade substrates occurred at the end of exponential phase. The strain CB001 degraded chlorobenzene with stoichiometric release of chloride ion. Activities of catechol 1,2-dioxygenase were higher than catechol 2,3-dioxygenase in Crude extracts from cells of strain CB001. It can be inferred that chlorbenzene was degraded through the pathway of meta ring cleavage. The strain NB001 degraded nitrobenzene with release of nitrite, but the ratio of amount of accumulated nitrite to the amount of degraded nitrobenzene. Activities of catechol 2,3- dioxygenase were higher than catechol 1,2- dioxygenase in Crude extracts from cells of strain NB001. It could be inferred that nitrobenzene was degraded through the pathway of ortho ring cleavage. The strain HL1 degraded hexachlorobutadiene and released only little amount of chloride ion. First order kinetics could be fitted to the biodegradation of chlorobenzene, nitrobenzene, and hexachlorobutadiene. The maximum of first order reaction rate constant was 0.0376 h^-1 for the biodegradation of chlorobenzne, 0.0330 h^-1 for nitrobenzene, and 0.0237 h^-1 for hexachlorobutadiene. The minimum of half-life for the biodegradation of chlorobenzene, nitrobenzene, and hexachlorobutadiene was 18.43 h, 21.00 h, and 29.24 h, respectively.(4) The promotion of degradability of strain HL1 by glucose was more than that of strain CB001 and NB001 by glucose. Cometabolism was an important mechanism for promotion of hexachlorobutadiene degradation by glucose. Glucose could increase the amount of released chloride ion but decrease the amount of released nitrite.(5) The degradation ability of strain CB001 on the compounds with similar structure to chlorobenzene were: 1,3-dichlorobenzene > l,2-dichlorobenzene> 1,4-dichlorobenzene > 1,2,3-trichlorobenzene > 1,2,4-trichlorobenzne > hexachlorobenzene. The degradation ability of strain NB001 on the compounds with similar structure to nitrobenzene were: 2-nitrotoluene >3-nitrotoluene >4-nitrotoluene >2,4-dinitrotoluene >2,6-dinitrotoluene. Strain HL1 showed strong biodegradability on trichloroethylene and tetrachloroethylene. Mixture of strain CB001 and NB001 showed biodegradability on chloronitrobenzenes, and the degradability was: 3-chlornitrobenzene>4-chloronitrobenzne>2-chloronitrobenzene.(6) Bio-membrane formed on the surface of gravels collected for Songhua River played a crucial role in the biodegradation of substrates in the gravel contact oxidation method simulation system, and the biodegradation ability of strain HL1 was the strongest among the strains. Strength of biodegradation of bio-membrane was enhanced with the increase of culture temperature, but after acclimated at lower temperature, the strains showed certain degradability. The microorganisms adsorbed on the surface of gravels and existing in the liquid phase promoted the biodegradation of substrates.(7) Sludge promoted greatly the biodegradation of substrates by strains in simulation system of microcosm with the most obvious promotion on the biodegradation of hexachlorobutadiene, and it was more obvious at early phase and last phase of biodegradation. Sunlight enhanced the degradation capability of strains to degrade substrates with the obvious promotion on biodegradation of substrates by strain CB001 and NB001. Decrease of substrate concentration in simulation of microcosm was mainly caused through the course of biodegradation, especially the degrading strains. Degrading strains could grow well in the simulation system of microcosm and existence of substrates could maintain the quantitative dominance, but the dominance could be maintained for a period of time through the feeding on other matters. Compared with other two strains, strain HL1 grew worse in simulation system of microcosm. Biodegradation rate of substrates by strains was increased with the increasing of culture temperature, but when acclimated at lower temperature and prolonged the culture time, biodegradation of substrates by strains could be found. Suspended particulates promoted the biodegradation of substrates by strains at early phase and the promotion could be maintained to the end of experiment for the biodegradation of hexachlorobutadiene, but the promotion was gradually depressed from the mid phase for the biodegradation of chlorobenzene and nitrobenzene. (8) Obvious inhibitory effect of Cd²⁺ and Hg²⁺ on the biodegradation of substrates by strains was observed at the concentration of 10.0 mg/L in two simulation system, and the inhibition of Hg²⁺ was greater than that of Cd²⁺. The addition of nutrient salts obviously enhanced the biodegradability. For the biodegradation of substrates in simulation system of gravel contact oxidation method, the promotion of nutrient salts was enhanced with the increasing of added amount, but for the simulation system of microcosm the trend of increase was stopped when the added amount was up to some degree. The promotion of nutrient salts on the degradation of hexachlorobutadiene by strain HL1 was the lowest.(9) Acclimated under certain procedures, strain CB001 showed satisfactory biodegradation on 1,3-dichlorobenzene, 1,2-dichlorobenzene, 1,4-dichlorobenzene which were coexisting in the two simulation system, strain NB001 showed lower biodegradability on 2-nitrotoluene, 3-nitrotoluene, 4-nitrotoluene, 2,4-dinitrotoluene , and 2,6-dinitrotoluen, strain HL1 showed strong biodegradability on trichloroethylene and tetrachloroethylene which were coexisting in two simulation systems. Mixture o strain CB001 and strain NB001 also showed biodegradability on chloronitrobenzenes. The biodegradability of strain CB001, NB001, and mixture of the two strains was stronger in simulation of microcosm than that in gravel contact oxidation method simulation system, but the opposite result was found for strain HL1. The order of biodegradability of mixture of strain CB001, NB001, and HL1 on the chlorinated hydrocarbons coexisting in two simulation system were chlorinated alkenes>chlorinated benzenes> chloronitrobenzenes>nitrobenznes.