Abstract: The role of broad-host range IncP-1 epsilon plasmids in the dissemination of antibiotic resistance in agricultural systems has not yet been investigated. These plasmids were detected in total DNA from all of 16 manure samples and in arable soil based on a novel 5'-nuclease assay for real-time PCR. A correlation between IncP-1 epsilon plasmid abundance and antibiotic usage was revealed. In a soil microcosm experiment the abundance of IncP-1 epsilon plasmids was significantly increased even 127 days after application of manure containing the antibiotic compound sulfadiazine, compared to soil receiving only manure, only sulfadiazine, or water. Fifty IncP-1 epsilon plasmids that were captured in E. coli CV601gfp from bacterial communities of manure and arable soil were characterized by PCR and hybridization. All plasmids carried class 1 integrons with highly varying sizes of the gene cassette region and the sul1 gene. Three IncP-1 epsilon plasmids captured from soil bacteria and one from manure were completely sequenced. The backbones were nearly identical to that of the previously described IncP-1 epsilon plasmid pKJK5. The plasmids differed mainly in the composition of a Tn402-like transposon carrying a class 1 integron with varying gene cassettes, IS 1326, and in three of the plasmids the tetracycline resistance transposon In 1721 with various truncations. Diverse Beta- and Gammaproteobacteria were revealed as hosts of one of the IncP-1 epsilon plasmids in soil microcosms. Our data suggest that IncP-1 epsilon plasmids are important vectors for horizontal transfer of antibiotic resistance in agricultural systems.

Abstract: The treatment of pig manure can be performed by anaerobic digestion to diminish the organic matter content and produce biogas, and the resulting digestate has to be subsequently treated for the removal of nitrogenous compounds. The partial nitritation-anammox (PN-AMX) process constitutes an interesting alternative. In the present study, three different short experiments were initially performed to study the influence of nitrite oxidizing bacteria (NOB) present in the inoculum and the pig manure composition over the start-up of the PN-AMX process. The presence of NOB in the inoculum showed to be more crucial than the available anammox activity for a good performance of the PN-AMX process. Batch activity experiments showed a reduction of at least 44.4% in the maximum specific anammox activity due to the pig manure, probably owed to its conductivity (between 6 and 8 mS/cm). In the subsequent long-term operation of the PN-AMX process with non-diluted pre-treated pig manure, the NOB were successfully limited for DO concentrations of 0.1 mg O-2/L, and a nitrogen removal rate (NRR) of 0.1 g N/(L.d) was achieved despite the presence of significant NOB activity in the start-up. A strict control of the DO concentration, with an optimal range of 0.07-0.10 mg O-2/L, was fundamental to balance the removal of nitrogen by PN-AMX and prevent NOB activity. The presence of organic matter, with a ratio sCOD/N in the influent between 0.18 and 1.14 g/g, did not hinder the PN-AMX process, and the contribution of heterotrophic denitrification to the removal of nitrogen was less than 10%.