Effects Of PAHs On Endophytic Bacteria In Ryegrass And Isolation, Performance Of A Phenanthrene-Degrading Endophytic Bacterium

Soil contamination has become a global environmental problem in recent decades. Polycyclic aromatic hydrocarbons (PAHs) are a class of common persistent organic pollutants in the soil environment with characteristics of teratogenic, carcinogenic and mutagenic, and can be absorbed and accumulated by plants and translocated into shoots, which is the major pathway for them to reach the food chain/web, resulting in serious threats to animal and human health. Clearly, obtaining the knowledge and ability to reduce the uptake of PAHs by plants will have considerable benefits in ecological risk assessments.Endophytic bacteria reside the internal tissues of healthy plants without causing apparent harm to their host. They are ubiquitous in roots, stems, and leaves of plants, and can benefit plant growth, increase plant tolerance, and promote degradative capacity. However, little literature is available on the effects of endophytic bacteria population characteristics on PAHs and the utility of PAH-degrading endophytic bacteria in reducing PAHs contamination. Hence, we seek to investigate whether the PAH-degrading endophtic bacteria would circumvent the risk of plant PAH contamination, protect the safety of agriculture products and the sustainable development of ecosystom.The article mainly studies the following aspects:(1) The effects of endophytic bacteria population characteristics on PAHs were detected using greenhouse hydroponic experiments; (2) Isolation and characteristics of an efficient phenanthrene-degrading endophytic bacterium strain from plants by enrichment culture. The objectives of this study were to elucidate the biology characteristics and degradation of phenanthrene by the isolated functional endophytic bacterium under different conditions, and provide a novel method of endophytic bacteria-aided cicumvention of the risk of plant PAH contamination. The main results as follows:(1) The ryegrasses absorb phenanthrene and pyrene from culture solution, the contaminations were removed from roots to stems, and with the increase of pollution concentration in culture solution, a significantly increase in the uptake of phenanthrene and pyrene by plants. Different PAHs pollution treatments of ryegrass contain rich diversity of endophytic bacteria. The cell counts of culturable endophytic bacteria in roots were significantly larger than stems and leaves. In the phenanthrene and pyrene combined pollution, with the decrease of phenanthrene concentration in culture solution, the cell counts of culturable endophytic bacteria in ryegrass were decreased, and high phenanthrene concentration caused culturable endophytic bacteria in ryegrass increased. Population of cultured endophytic bacteria and advantage species in ryegrass can be changed by different PAHs pollution. Based on morphological, physical and chemical properties and 16S rRNA gene analysis,33 kinds of culturable endophytic bacteria were isolated from different phenanthrene and pyrene contaminated ryegrass, belonging to 7 classes:Gamma Proteobacteria、Sphingobacteria、Alpha Proteobacteria、Bacilli、Flavobacteria、Beta Proteobacteria、Actinobacteria. Some of them were able to degrade phenanthrene or pyrene by using it as the sole carbon source.(2) In this study,10 phenanthrene tolerant endophytic bacterial strains were isolated from plants grown in PAH-contaminated soils, and a high-efficiency phenanthrene-degrading endophytic bacterial strain PHE-3 was isolated from healthy plant Plantago Asitica Linn.. Strain PHE-3 can use phenanthrene as the sole carbon source for growth and the degradation of phenanthrene. Based on the morphology, physical, chemical properties and 16S rRNA gene sequence analysis, strain PHE-3 was identified as Paenibacillus sp.. Strain PHE-3 had diauxic growth characteristics in LB liquid medium. The phenanthrene degradion of strain PHE-3 up to 99% in 96 h in phenanthrene-degrading culture, and the number of bacterial strain reached the maximum. Moreover, the resistance tests revealed that strain PHE-3 was sensitive to a variety of antibiotics, but was resistant to low concentration of ampicillin.(3) The degradation and characteristics of phenanthrene by strain PHE-3 with different environmental factors were investigated. It was observed that 99% of phenanthrene was degraded by PHE-3 at 30℃ and 150 r·min=1 in 84 h under the condition of pH 4.0-8.0. Strain PHE-3 had a better effect on the degradation of phenanthrene at 30-42 ℃, which the rates of phenanthrene degradation is all above 90%. When the temperature is low (20℃), the degradation rates of phenanthrene by strain PHE-3 is only 19%. Different substrates concentration on the degradation of phenanthrene by strain PHE-3 has different effects, with increasing phenanthrene concentration, the rates of phenanthrene degradation was decreased by strain PHE-3. The addition of exotic carbon and nitrogen sources in medium could effectively enhance phenanthrene degradation. When the addition of exotic carbon source is citric acid/ammonium sulfate, nitrogen source is ammonium nitrate, or additional mixture of carbon and nitrogen sources are glutamic acid and yeast, phenanthrene could be degraded in 84 h by strain PHE-3. Strain PHE-3 also has degradion of other PAHs, such as naphthalene, fluorine, pyrene, fluoranthene or benzo[a]pyrene to a certain extent.