Isolation, Characterization And Bioaugmentation Study Of An Efficient O-Nitrobenzaldehyde Degrading Strain Pseudomonas Putida ONBA-17

Along with the industrialization advancement speeding up in China, the wastewater discharged by organic synthesis industry is more and more huge, which has caused serious pollution to the environment and threatened the life and health of human. O-nitrobenzaldehyde （ONBA） is used as an important intermediate for the synthesis of pharmaceuticals, dyes, agrochemicals, and other organic compounds. In the southeast of China, at least 730,000 tons of ONBA were produced per year. What’s more, vast quantities of wastewaters containing ONBA were generated during the manufacturing process. Because of its toxic and recalcitrant nature, ONBA creates pollution hazards. In order to uncover its harmful nature, supply certain design and operation information and experience to relevant activated sludge mediated wastewater processing actions, meanwhile study the feasibility of its bioaugmentation and changes in sludge composition and microbial community, and isolation and characterization of predominant population, etc, I carried out this research.I initially studied the characteristics and components of the wastewater, the high CODcr value, suspension solids content and salinity was confirmed; 2-ethylhexanol, ONBA, o-nitrobenzenemethanol, dibutyl phthalate and diisooctyl phthalate was indeed presented in the aqueous phase through GC-MS analysis. Besides, through the acute toxicity test analysis, the LD₅₀ value of ONBA is in the range of medium toxicity.Then, sets of sequencing batch reactors （SBR） were built up in our laboratory. Hydraulic retention time and sludge retention time were respectively studied as variables, and the relationship between biomass of activated sludge and the removal rates of COD_Cr and total nitrogen was examined. The model of wastewater treatment under such conditions was built up as S_e = K_s （1 + b·θc） / [θ_c（Y·K - b） - 1] = 996（1 + 0.1970c） / （0.730c - 1） and X = r （S₀-S_e）·θc/θ_H （1+b·θc） = 0.342（S₀ - S_e）·θ_c /θ_H （1 + 0.197·θ_c）. Through the investigation around changes in activated sludges, I found that the settling ability of the adapted sludge was improved as compared with the seeding sludge. Further study revealed that this improvement was relevant with the drop in protein/carbohydrate ratio of sludge extracellular polymeric substances; the sludge structure gradually became compact and the microorganism community also changed remarkably. Combined with the methods of fluorescent in situ hybridization （FISH） and denaturing gradient gel electrophoresis （DGGE）, changes in bacterial community of sludge were thoroughly studied, and predominant bacterial populations in adapted sludge were identified as populations which belong to the a- andβ-Proteobacteria. Microbial ecology study carried out in this way might overcome some bias and be more comprehensive and persuasive as compared with the single-method studied one.In order to realize the specific biodegradation of ONBA and supply proper strain candidates for bioaugmentaion, I initiated the screen and isolation of ONBA-degrading strain which used activated sludge sampled form a domestic wastewater treatment plant as innoculum. Several bacterial strains which could utilize ONBA were obtained. A strain, designated as ONBA-17, was used for further study, because it could completely degrade 100 mg/mL ONBA in 12 h and owns some other aromatic compounds degrading ability. The optimum growth conditions were 28"C and pH 7.6; fructose and yeast extract were its optimum carbon and nitrogen sources, respectively. The isolate was a moderately halophilic bacterium due to its optimal growth salinity was 3.5%, and multiresistant to some heavy metals and antibiotics.Through detailed study on the incubation, physiological and biochemical characteristics of the strain, and analyses of its 16S rDNA sequence, G+C mol% content and DNA-DNA hybridization, this strain was identified as Pseudornonas putida species and named Pseudornonas putida ONBA-17. Afterwards, the optimal effect conditions of crude enzymatic extract were 30℃and pH 7.4. Besides, the effects of some metal ions, EDTA and surfacants on the extract were evaluated. The result indicates that Li⁺, Ni²⁺ and Co⁴⁺ have inhibitory effect on it, but Ca²⁺, Mg²⁺ and Mn²⁺ could activated it. Tween 20, Tween 80 and especially SDS also inhibit the enzyme（s） very much. The fact that EDTA could reduce approximately half of the enzyme activity implied that the enzyme（s） might belong to one kind（s） of the metalloenzyme; In addition, I studied ONBA-degrading metabolism pathway and localization of the relevant gene（s）. These genes might be existed on the chromosome of the strain.Through tri-parental mating, a fragment of extraneous source gfp gene was inserted into the chromosome of P. putida ONBA-17. The stability of it was confirmed by successive culture either on solid or in liquid LB medium. During the process of evaluating the effect of bioaugmentation, I found that bioaugmentation not only enhanced the removal effect of the target compound, but also significantly shortened the system start-up time; however, it was ephemeral. Our study supported the idea that natural uncontaminated environmental systems contain sufficient genetic diversity to make them valid choices for the removal of xenobiotics after an adequate exposure time, either by metabolism or co-metabolism.Through confocal laser scanning microscopy （CLSM） observation, I on-line tracked the strain. Our observations suggested that predation by protozoa is a major cause for the disappearance of the introduced bacteria. Further, we considered that the fate of the inoculum species mainly depends on three factors, which are, the adaptation and retention capabilities of the strains, the competition between the allochthonous bacteria and the indigenes, and the capability against predatory protozoa.In order to study the influence caused by bioaugmentation on microbial community and supply some guidance for later isolation of functional dominant population, we applied DGGE technique to monitor changes in reactor systems. Then, we found that bioaugmentation itself as compared with the operating conditions and environment exerted less influence on microbial community structure. A part of a- andβ-Proteobacteria, and certain unknown groups gradually became the dominant populations.Through plate spreading I obtain four bacterial strains, and then identified and characterized them. The result shows that these strains, designated as R, S, W and M respectively, were identified as Exiguobacterium sp., Pseudomonas sp., Bacillus cereus and P. aeruginosa. A systematic study has been conducted to identify the ONBA-degrading pairs among isolates and strain ONBA-17, but no positive combination was found. I further checked their surface hydrophobicities and aggregation abilities; interestingly all of them are good floc formers. However, none of them could utilize ONBA. Combine with the operation characteristics of SBR system, why bacteria own such traits could be kept and screened out could be explained to some extent. What’s more, the partial 16S rDNA sequences of them were compared with the sequences of the dominant populations in the activated sludge that were detected by the DGGE analysis. This comparison revealed that strain M and S are very closely related to some DGGE bands indicating bacteria. Finally, through comparing main carbon source utilization degree and GC-MS analysis, the phenomenon of growth promotion could be explained to some extent. Best to our known, it’s the first systematic research on aspects of ONBA-degrading strain isolation and relevant bioaugmentation, etc. what’s more, there was no such wastewater treatment model in domestic published studies. Therefore, it owns certain originalities, and could laid scientific foundations and gave some hints, such as better understanding of the distribution of metabolic functions within multi-species communities and how these microhabitats in turn affect community architecture, for future researches.