Biodegradation Of Phenanthrene By Klebsiella Sp.ZS1 Under Low-oxygen Condition

Low-oxygen condition, the main accumulation place of organic pollutants, widely existed in all types of water, sediments and soil. Due to the lack of oxygen in the hypoxic environment, biodegradation of pollutants in sediment and water was likely to be carried out under an mixed electron acceptor environment. Compared with the aerobic degradation and anaerobic degradation, microbial degradation of PAHs under low-oxygen condition was far from enough. First, the effects of different electron acceptors on the degradation of polycyclic aromatic hydrocarbons(PAHs) were still lack of research; there was no research on the intermediate products and pathways of PAHs biodegradation. Therefore, PAHs degrading bacteria, had been screened under low-oxygen condition, were identified and one of them was selected for further research. The degradation of phenanthrene effected by different electron acceptors was investigated and its hypoxia degradation pathway was explored. The main methods and conclusions of this study were as follows:(1) Nine PAHs degrading bacteria were identified by scanning electron microscopy, 16 S rDNA sequence analysis and phylogenetic trees. Phe-1 strain was belong to Stenotrophomonas sp.; Phe-3, Phe-4, Phe-5, Phe-6 strains were belong to Klebsiella sp.; Pyr-2 and Pyr-3 were belong to the genus Bacillus sp.; Pyr-4 was belong to Raoultella sp.; Pyr-6 was belong to Comamonas sp.. Studies of PAHs degrading bacteria both at home and abroad indicated that Klebsiella sp. and Bacillus sp. were very common in PAHs degradation and played an important role in the natural repair of PAHs.(2)In order to investigate the effects of different electron acceptors on the degradation of polycyclic aromatic hydrocarbons(PAHs) by Klebsiella sp. ZS1 under low-oxygen conditions, 10 mg/L phenanthrene was used as the sole carbon source, and a PAH-degrading bacterial strain(Klebsiella sp. ZS1) was inoculated, the biodegradation ability was test with the addition of three common electron acceptors, namely, 20 mmol/L Na2SO4, 20 mmol/L NaNO3, 10 mmol/L FeCl3, respectively. Low-oxygen condition(8% O2) was controlled in a hypoxia station. The electron acceptor utilization and bacterial growth were measured by the national standard analytical methods; the remaining phenanthrene was determined by gas chromatography–mass spectrometry(GC–MS);changes of the bacterial growth and the remaining phenanthrene were analyzed by one-way ANOVA, respectively. The results showed that, Klebsiella sp. ZS1 used SO42-, NO3- and Phenanthrene only was decreased by 10.1% without inoculating Klebsiella sp. ZS1. However, when Klebsiella sp. ZS1 was inoculated, the degradation rates of no addition of electron acceptor, addition of SO42-, NO3- and Fe3+ were 68.9%, 86.2%, 72.9% and 68.9%, respectively; the degradation rate constants determined by using a first-order kinetic equation were 0.181 d-1, 0.360 d-1, 0.186 d-1, 0.183 d-1 with the correlation coefficients were of 0.986, 0.992, 0.966 and 0.943, respectively. The bacteria growth of SO42- addition group was 2.5 times compared with the control group, while the groups of addition NO3- and Fe3+ were near to the control, respectively. The changes of the bacterial growth and the remaining phenanthrene had no statistically significant difference between the three groups, namely, addition of NO3-, Fe3+or without any electron acceptor addition, respectively. While the addition of SO42- had significant difference with other three groups. The results indicated that, Klebsiella sp. ZS1 can use SO42-, O2 at the same time, as its electron acceptor under low-oxygen condition, while NO3- and Fe3+ can not be used. Furthermore, addition of SO42- as electron acceptor obviously stimulated the biodegradation of phenanthrene and the growth of the strain when compared to the control. However, the addition of NO3- and Fe3+ had no statistically significant effect on the reduction of phenanthrene, neither the growth of the bacterium.(3) In order to explore intermediates and degradation pathway of phenanthrene degradated by Klebsiella sp. ZS1 under 8% oxygen partial pressure conditions, 10 mg/L deuterium-labeled phenanthrene was used as the sole carbon source and a PAH-degrading bacterial strain(Klebsiella sp. ZS1) was inoculated without any exogenous electron acceptors addition. The degradation products were divided into acidic and neutral intermediates by extraction with ethyl acetate and 10 mmol/L NaOH. The acidic products wrer analyzed by GC-MS after MTBSTFA derivatization. The neutral intermediates wrer also analyzed by GC-MS after concentration.Analysises of each substance peaks determined the most probable molecular formula and structure. Changes of chromatogram peak area over time revealed the accumulation or depletion of intermediates.Based on the above analysis, the degradation pathway of PAHs was speculated as follows: under hypoxic conditions, degradation of phenanthrene by Klebsiella sp. existed characteristics of both aerobic degradation and anaerobic degradation. First, the dioxygenase or monooxygenase make an aromatic ring hydroxylation in phenanthrene; a carboxy-substituted hydrogen on the aromatic ring at the same time. Then, lipase produced by Klebsiella sp. ZS1 contributed to esterification reaction, namely dehydration of hydroxyl and carboxyl groups formed an ester; this process was accompanied by cracking of phenyl ring. Finally, a part of material were no longer decomposed and stayed in the culture medium, the other part further broken into smaller molecular weight molecules.