Performance And Mechanismes Involved In Reducing Pyrene Pollution In Water Spinach With Pyrene-Degrading Bacterium Mycolicibacterium Sp. Pyr9

Polycyclic aromatic hydrocarbons（PAHs）are a type of representative refractory organic pollutants that are ubiquitous in the environments.They have chronic toxicity and potencial carcinogenic,teratogenic,and mutagenic effects,posing a certain threat to the health of animals,plants and humans.Studies have shown that PAHs in the soil can be absorbed by plants and further enriched through the food chain,threatening the health of human population.Therefore,how to reduce the PAH pollution of crops in polluted areas has become a problem that needs to be solved urgently.Recent studies have shown that there are PAH-degrading bacteria in inner tissue and root surface of healthy plants grown in PAH-contaminated areas.Isolating these bacteria and recolonizing them in plants can effectively reduce the absorption and accumulation of PAHs by plants,thereby reducing the risk of plant PAH pollution in these sites.This technology is of high efficiency,environmental friendliness and low cost.However,the current researches mostly focus on endophytic bacteria in plants and low molecular weight（LMW）PAHs,there are few reports about utilizing root-associated PAH-degrading bacteria to prevent the absorption of high molecular weight（HMW）PAHs in the rhizosphere by plants,and the mechanism involved is also unclear.Based on this,in this study,with the pyrene as the representative HMW PAHs,the pyrene-degrading bacterium,Mycolicibacterium sp.Pyr9,isolated from the root surface of healthy plant Eleusine indica L.Gaertn.in PAH-contaminated area,as the target strain,and water spinach（Ipomoea aquatica Forssk.）as the host plant,the performance and mechanism of using root-associated pyrene-degrading bacterium to reduce plant pyrene pollution were then systematically studied.The main results are shown as follows:1.The biological and pyrene-degrading characteristics of strain Pyr9 were clarified,the key genes responsible for pyrene degradation in strain Pyr9 were analyzed.The root-associated bacterium Mycolicibacterium sp.Pyr9 could effectively reduce the pyrene in MSM culture,and the degradation rate of pyrene reached more than 98%within 8days(the initial pyrene concentration is 50 mg·L^-1).The metabolic intermediate products of pyrene degraded by strain Pyr9 were analyzed by high performance liquid chromatography（HPLC）combined with mass spectrometry（MS）,according to the the reported literature.The results indicated that strain Pyr9 should metabolize pyrene via the phthalic acid pathway.Meanwhile,strain Pyr9 was capable of growing with a variety of PAHs as the sole carbon source and energy source,and it also showed a good degradation abilty to low-ring PAHs such as acenaphthylene,acenaphthylene and phenanthrene.In addition,strain has the ability to form stable biofilms on R₂A medium,and owns a variety of plant growth-promoting properties.Strain Pyr9 could dissolve organic and inorganic phosphorus effectively,but its ability to secrete IAA,produce siderophores,dissolve potassium and fix nitrogen was relatively weak.The key genes responsible for pyrene degradation derived from Mycolicibacterium sp.in database of National Center for Biotechnology Information were analyzed,and it was found that most of these genes were highly conserved.After primers designing based on the sequence of the conserved region,the target genes were amplified using PCR and sequenced,six key genes responsible for pyrene degradation（nid A,nid B,nid C,nid D,nid H and nid X gene,respectively）were identified from strain Pyr9.The fluorescence quantitative PCR（q PCR）technology was further used to monitor the transcription levels of the six key genes in strain Pyr9 under pyrene induction,and it was found that the transcription levels of these six genes were significantly up-regulated under pyrene induction compared with the control group without pyrene,but their peak transcription level and changing trend with induction time were not synchronized.This phenomenon indicated that the transcription of the six key genes responsible for pyrene degradation in strain Pyr9 will be regulated by pyrene,however,since the encoding enzymes involved in were different,their changing trends were also different.2.The colonization,distribution and performance of strain Pyr9 in water spinach-solution system were explored.Strain Pyr9 was colonized on the root surface of water spinach by soaking roots before greenhouse hydroponic experiments,and its effect on the absorption and accumulation of pyrene in water spinach were investigated.The results showed that strain Pyr9 could effectively colonize on the root surface of water spinach and further migrate to the shoots through root tissues,and the cell counts of Pyr9 on root surface was the highest.Meanwhile,compared with Pyr9-free treatment,the pyrene contents in the roots and shoots of water spinach and in the culture solution were much lower than those in the Pyr9-inoculated treatment.Correspondingly,the pyrene accumulation in the roots and shoos of water spinach were also reduced.For instance,on the 6th day,compared with that in Pyr9-free group,the pyrene content in the shoots of water spinach in Pyr9-inoculated group was reduced by 76%（S1）,69%（S2）and 78%（S3）,respectively.After 15 days of hydroponics,compared with those in Pyr9-free groups,the pyrene contents in the roots of water spinach in Pyr9-inoculated groups were all reduced by more than 85%.Obviously,the colonization of strain Pyr9 could effectively reduce the absorption and accumulation of pyrene by water spinach under pyrene-polluted conditions.3.The mechanisms involved in reducing pyrene contamination of water spinach by colonization of strain Pyr9 were calarified.The gene abundance of nid A（coding for the large subunit of initial hydroxylated dioxygenase）in the water spinach-solution system was determined through absolute quantitative PCR technique.The results suggested that the colonization of strain Pyr9 in water spinach enhanced the nid A abundance significantlyin this system.The abundance of nid A in the water spinach-solution system was higher in root than in stem and leaf than in solution,and the nid A abundance in the Pyr9-inoculated treatment groups were significantly higher than those in the Pyr9-free treatment groups.The highest gene abundance of nid A in roots,shoots and culture solution of Pyr9-inoculated groups was7.11±0.07 Log g^-1 d.w.s,5.88±0.42 Log g^-1 d.w.s and 3.85±0.01 Log m L^-1,respectively;while that in Pyr9-free groups was only 4.93±0.03 Log g^-1 d.w.s,3.99±0.10 Log g^-1 d.w.s and 2.58±0.32 Log m L^-1,respectively.In addition,through high-throughput sequencing analysis of the 16S r RNA gene,it was found that the Pyr9 colonization changed the bacterial community structure in water spinach–solution system,and the relative abundance of Mycobacterium sp.,which may take part in the pyrene degradation,was significantly increased.It is speculated that Pyr9 promoted the formation of the interaction network of pyrene metabolism flora in the water spinach,accelerated the degradation of pyrene in the polluted environment.