[Effect of Nitrate Amendment on Soil Denitrification Activity and Anthracene Anaerobic Degradation].

The degradation of soil polycyclic aromatic hydrocarbons (PAHs) under denitrification is one of the most important pathways for anaerobic PAH elimination, but little is known about the effect of nitrate (the terminal electron acceptor for denitrification) on soil denitrification activity and PAH degradation under anaerobic conditions. In this study, the effect of nitrate on soil anthracene anaerobic degradation and denitrification activity was investigated through an anaerobic microcosm experiment. Two groups of treatments without (N0) and with (N30) nitrate (30 mg·kg-1) amendment were conducted. Each group contained three treatments with different anthracene concentrations (0, 15, and 30 mg·kg-1, denoted as A0, A15, and A30, respectively). Therefore, a total of six treatments (N0A0, N0A15, N0A30, N30A0, N30A15, and N30A30) were incubated in darkness at 25℃ for 45 days, and the production rates of N2O and CO2, abundances of denitrification related genes (narG:periplasmic nitrate reductase gene; nirK:copper-containing nitrite reductase gene; and nirS:cd1-nitrite reductase gene), and soil anthracene content were measured at 3, 7, 14, and 45 days. The results indicated that the intensive denitrification enzyme activity in each treatment was only detected at day 3, which could be significantly enhanced by both nitrate and anthracene amendments. Subsequently, a sharp decline of denitrification enzyme activity was observed in each treatment, while anthracene showed an obvious inhibition of soil denitrification enzyme activity. The result of a two-way ANOVA also indicated that nitrate, anthracene, and their interactions had significant effects on soil denitrification enzyme activity. The result of a quantitative-PCR indicated that, during the incubation, the abundances of narG and nirS exhibited an increasing tendency, but the abundance of nirK was relatively constant compared with its former counterparts. The final removal rate of anthracene under anaerobic soil environment was in the range of 33.83%-55.01%, and neither the final removal rate nor the degradation rate of anthracene could be significantly affected by nitrate amendment during incubation. The anthracene degradation rates in the higher anthracene containing treatments (N0A30 and N30A30) were significantly higher than those in the lower anthracene containing treatments (N0A15 and N30A15). In summary, nitrate amendments had no effect on soil anthracene anaerobic degradation but could significantly affect soil denitrification enzyme activity and the abundance of denitrification related narG and nirS genes.

Co-effects of pyrene and nitrate on the activity and abundance of soil denitrifiers under anaerobic condition.

It has previously been confirmed that polycyclic aromatic hydrocarbons (PAHs) could be degraded by soil microbes coupling with denitrification, but the relationships among soil denitrifiers, PAHs, and nitrate under obligate anaerobic condition are still unclear. Here, co-effects of pyrene and nitrate on the activity and abundance of soil denitrifiers were investigated through a 45-day incubation experiment. Two groups of soil treatments with (N30) and without (N0) nitrate (30 mg kg-1 dry soil) amendment were conducted, and each group contained three treatments with different pyrene concentrations (0, 30, and 60 mg kg-1 dry soil denoted as P0, P30, and P60, respectively). The pyrene content, abundances of denitrification concerning genes (narG, periplasmic nitrate reductase gene; nirS, cd 1-nitrite reductase gene; nirK, copper-containing nitrite reductase gene), and productions of N2O and CO2 were measured at day 3, 14, 28, and 45, and the bacterial community structures in four represented treatments (N0P0, N0P60, N30P0, and N30P60) were analyzed at day 45. The results indicated that the treatments with higher pyrene concentration had higher final pyrene removal rates than the treatments with lower pyrene concentration. Additionally, intensive emission of N2O was detected in all treatments only at day 3, but a continuous production of CO2 was measured in each treatment during the incubation. Nitrate amendment could enhance the activity of soil denitrifiers, and be helpful for soil microbes to sustain their activity. While pyrene seemed had no influence on the productions of N2O and CO2, and amendment with pyrene or nitrate both had no obvious effect on abundances of denitrification concerning genes. Furthermore, it was nitrate but not pyrene had an obvious influence on the community structure of soil bacteria. These results revealed that, under anaerobic condition, the activity and abundance of soil denitrifiers both were insensitive to pyrene, but nitrate could improve the activity of soil denitrfiers and induce the shifts in soil bacterial community structure.

Comparative Investigation of Bacterial, Fungal, and Archaeal Community Structures in Soils in a Typical Oilfield in Jianghan, China.

Agricultural soils in oilfields have high risk for polycyclic aromatic hydrocarbon (PAH) pollution. In this study, from the Jianghan Oilfield (Hubei Province, China) with a history of >50 years, 7 soil samples (OS-1 to OS-7) were collected. Subsequently, the bacterial, archaeal, and fungal community structures were investigated by Illumina MiSeq sequencing, and the relationship between microbial community structure and soil PAH content was analyzed. The results indicated that bacterial and archaeal Chao 1 indices showed a significantly negative relationship with soil PAH content, and only the bacterial Shannon index had a significantly negative relationship with soil PAH content. Moreover, the community structure of bacteria (r 2 = 0.9001, p = 0.013) showed a stronger correlation with PAH content than that of fungi (r 2 = 0.7357, p = 0.045), and no significant relationship was found between archaeal community structure (r 2 = 0.4553, p = 0.262) and soil PAH content. In addition, the relative greater abundances of some bacterial genus belonging to Actinobacteria (Mycobacterium and Micromonospora) and Proteobacteria (Pseudomonas, Lysobacter, Idiomarina, Oxalobacteraceae, and Massilia), fungal genus belonging to Ascomycota (Sordariales and Pleosporales), and archaeal phylum (Euryarchaeota) were detected in the soil samples (OS-3 and OS-5) with greater PAH content. In summary, soil PAHs showed an obvious influence and selectivity on the soil microbiota. Furthermore, compared with fungi and archaea, bacteria was more sensitive to soil PAH pollution, and the diversity indices and community structure of bacteria both might be suitable indicators for assessment of soil PAH stress on the soil ecosystem.

[Effects of PAHs Pollution on the Community Structure of Denitrifiers in a Typical Oilfield].

Agricultural soils in the oilfields have the potential risk of PAHs (polycyclic aromatic hydrocarbons) pollution, and the denitrification process with nitrate as the terminal electron acceptor might be important for soil PAHs elimination under anaerobic condition. In this study, 9 soil samples listed as JH-1 to JH-9 were collected from the JiangHan oilfield with a history of more than 50 years. Using the functional genes (nirK: Cu-nitrite reductase gene; nirS: cd1-nitrite reductase gene) involved in denitrification as biomarkers, the community structure of soil denitrifiers was investigated by quantitative-PCR and T-RFLP (terminal-restriction fragment length polymorphism) combined with clone library, and the relationship between soil properties and community structure of soil denitrifers was discussed. The result indicated that the copy numbers of nirK were higher than those of nirS in all soil samples, and the lowest copy numbers of nirK and nirS were both detected in the JH-4 with the highest PAHs content. Meanwhile, the correlation analysis also showed a negative correlation between the copy numbers of those functional genes and soil PAHs content (nirK: R2=0.54, P<0.05; nirS: R2=0.58, P<0.05). Furthermore, the result of T-RFLP indicated that the nirK community structures in different soil samples varied significantly, which was obviously unique in the sample (JH-4) with the highest PAHs content. The subsequent RDA (redundancy analysis) also demonstrated that soil PAHs content as well as the available nitrogen and phosphorus belonged to the most important factors affecting the nirK community structure in this oilfield soil. Compared with nirK, little variation was shown about the nirS community structure among the soil samples. However, the abundance of nirS-harboring pseudomonas had a remarkably positive relation with the soil PAHs content, which indicated that pseudomonas, a well known bacterial genus with strong ability to degrade organic pollutants, might be an essential driver for PAHs degradation via denitrification process in this oilfield soil.