Isolation, Degradation Characteristics And Metabolic Pathways Of Pyridine-degrading Strain From Activated Sludge From A Coking Wastewater Treatment

Nitrogen heterocyclic compounds （NHCs） are the main organic contamination in coking wastewater. The concent of NHCs is about 20-30% of total organic compounds and their total concentration exceeds 200 mg/L. Most NHCs with malodor and toxicity pose a threat to the environment and human health, and provide inhibition effects on microorganism activities. More than 90% of HNCs was found to be removed during the aerobic stage of coking wastewater treatment process, which indicates activated sludge domesticated by the complex pollutants in coking wastewater may contain diverse bacteria with versatile degrading characteristis. The obtainment and investigation of versatile degrading bacteria will contribute to improve the removal efficiencies of NHCs from wastewater in biological treatment, and decrease environmental pollution of NHCs. In the present work, using pyridine and quinoline as model componds of NHCs, the NHCs-degrading bacteria were isolated from the activated slude of a coking wastewater treatment. The degradation characterstics under various growth and nutrition condition, the growth kinetics and metabolic pathways of the isolated were investigated. The quantitative structure biodegradation relationships （QSBR） of NHCs were also studied. The conculsions are as follows:（1） A pyridine-degading bacterium species （named as DN-06） was novely isolated from the aerobic activated sludge in coking wastewater treatment plant. DN-06 was identif ied as Achromobacter sp. using 16S rDNA sequence analysis. The optimal growth conditions are: pH 7-8, 30-35°C and shaing speed 150-170 r/min. The effects of different nutiution condition including various metal ions, nitrogen and carbon source on pyridine biodegradation by DN-06 were investagted. （I） Fe³⁺, Mn²⁺ and Zn²⁺ facilitate pyridine degradation, while Mo（VI） and Cu²⁺ provide inhibition effect. （II） Urea （< 36 mg/L） and ammonia （< 200 mg/L） do not affect pyridine degradation. Nitrite inhibites distinctly pyridine degradation by DN-06. Low concentration of nitrate （< 30 mg/L） has negligible effect on pyridine degradation, but high concentration of nitrate （50-80 mg/L） will inhibit pyridine degradation. （III） Low concentration of glucose can accelerate DN-06 growth and pyridine degradation. DN-06 can utilize both substrates in the double-substrate experiments, such as phenol+pyridine, and pyridine+quinoline. Antagonistic effect was found between the substrates, and the effects on pyridine by phenol and quinoline are larger than effects from pyridine.（2） The growth kinetics of DN-06 follows inhibition kinetic quation. DN-06 utilizes pyridine in a wide concentratin from 50-4300 mg/L. As pyridine concentration increases, the lag period is prolong and the specific growth rate （μ） increases first and then edcreases. Five models （i.e. Haldane, Yano, Aiba, Webb and monod model） were used to fitted by non-linear regression on S0 andμ. Rusults indicate that Haldane and Yano kinetic models are more suitable models for full substrate concentration range with the highest R2 values of 0.929. R2 = 0.929. The growth of Achromobacter sp. DN-06 follows substrate inhibition kinetic model. The kinetic parameters ofμmax, Ks and Ki in Haldane model are 0.161 h^{- 1}, 142.6 mg /L and 4234.5 mg /L, respectively. The high values ofμmax and Ks indicate that Achromobacter sp. DN-06 has a good tolerance against high pyridine concentrations.（3） The metabolic pathways of pyridine and quinoline by DN-06 were investigated using intermediary metabolite detection by UV spectroscopy, HPLC and GC/MS, and assays of enzyme activities. The metabolic pathways of pyridine in Achromobacter sp. DN-06 likely involve the direct cleavages of the ring via both the N-C2 and C2-C3 cleavage pathways. The metabolic pathway of quinoline by DN-06 was proposed, that is, via 8-hydroxycoumarin pathway, since metabolic intermediates were identified, including 2-hydroxyquinoline, 2,3-dihydroxyphenylpropione and 8-hydroxycoumarin. Achromobacter sp.DN-06 is the newly reported pyridine and quinoline-degraing bacterium. The metabolic pathways of pyridine and quinoline provide a supplement for NHCs degradation.（4） A tri-wavelength UV/Vis spectrophotometric method for rapid determination of quinoline （Q） and 2-hydroxyquinoline （HQ） during Q biodegradation was developed on the base of the spectral measurements at 289 （the isosbestic point of Q and HQ）, 326, and 380 nm（EPS）. The spectral interference of extracellular polymeric substances （EPS） in the process samples could be minimized, and the amounts of Q and HQ could be simultaneously quantified.The present method is simple, rapid, and suitable for the investigation in Q biodegradation processes.（5） A correlation between chemical structures of NHCs and their biodegradation activities was found. Mainly based on some quantum chemical descriptors computed at the B3LYP/6-311+G（d） level with Gaussian software, and the manually calculated molecular connectivity indexes（MCI）, by the use of least squares analysis, a quantitative structure-biodegradation relationship （QSBR） model for biodegradation rates of selected 7 NHCs were obtained.The correlation coefficients of the obtained models are significant. The results showed that the eigenvalue of the highest occupied molecular orbital（ELUMO–EHOMO）, mulliken atomic charges on the most negative hydrogen atom（Q_H^–）, and trialence MCI（⁴X^V_P ） play an important role in governing the biodegradation of NHCs.The results indicate that bacteria can obtain specific degradation characteristics under pollutants pressure and long-term domestication in coking wastewater, and the activated sludge in coking wastewater treatment opens the door for the source of functional bacteria.