Horizontal Transfer Of Resistant Plasmid RP4 In Different Microbial Aggregates And Its Effect On Ammonia Oxidation

The transmissions of antibiotic resistance genes(ARGs) severely threat to human health. As a class of emerging environmental contaminants, ARGs have drawn increasing attention from researchers worldwide. World Health Organization reported that ARGs will be one of the most significant challenges to human health in this century. BBC reported that the death toll of antibiotic-resistant infection would exceed that of cancer after 2050. As an important intermediary, aquatic environment can provide favorable conditions for rapid spread of ARGs, and has become a repository of ARGs. The biological wastewater treatment system is not only an important pathway of antibiotics entering the environment, but also an important source of pollution that enrich and spread ARB and ARGs. The horizontal transfer of ARGs in biological treatment system is directly related to the environmental and ecological safety.Biological wastewater treatment system is complicated. Due to the different operating modes and the process of construction of the reactor, different forms of microbial aggregates can be formed. In this study, a lab-scale granular sludge bioreactor(GSBR) where flocculent and granular sludge coexisted was designed. The particle diameter(D) < 0.18 mm defined as flocculent sludge. Granular sludge is divided into three ranges: the small size aggregates(0.18 mm < D <0.45mm), the moderate size aggregates(0.45 mm <D <0.9mm) and largest aggregates(D > 0.9mm). The different microbial aggregates were taken as objects and real-time quantitative PCR was applied to investigate ARGs transfer frequency and its regular pattern in different microbial aggregates. The results show that after the inculation of E.coli K12(RP4) on day 7, the aggregates size distribution changed, and then tended towards stability after day 18. However throughout the experiment, over 70% of the biomass was the largest aggregates, so the largest granular sludge was the main microbial aggregates form in GSBR. After the inculation of E.coli K12(RP4), with the donor strain being washed out, RP4 decreased in GSBR. As the size of aggregates increased, the abundance of RP4 in sludge reduced. The largest aggregates(D>0.9mm) contained the least RP4 plasmid which was undetected after 7 days. The RP4 plasmid was not detected in the aggregates of sizes(0.45mm<D<0.9mm, 0.18 mm<D<0.45 mm) after 14 and 13 days. For the flocculent sludge, the ratio of RP4 copies to 16 s rDNA remained relatively stable at 1.2×10-6-1.5×10-6 for 18 days then the RP4 plasmid was undetected. Therefore, using granular sludge system may reduce the transmissions of ARGs and lessen potential ecological threats, thereby improving the ecological safety of water treatment processes. With the absence of antibiotic selection pressure, RP4 remained stable in flocculent sludge for a long time, which still had an impact on the ecological system. ARGs have exceeded the scope of antibiotics pollution. To control the risk of ARGs, on one hand reducing the use of antibiotics is needed, on the other hand, controlling the transfer and dissemination of ARGs is significant.Nitrification is important in the nitrogen cycle in nature environment, and is critical in wastewater biological denitrification system. Nitrification involves two steps: ammonia is oxidated to nitrite and nitrite is oxidated to nitrate. Ammonia oxidation by ammonia oxidizing bacteria(AOB) is the rate-limiting step in nitrification, and AOB are more sensitive to environmental factors. ARGs, as biological macromolecules, can be involved in the bacterial metabolism, and even affect, alter biological metabolic processes, and thus directly affect the removal efficiency of biological wastewater treatment systems. This report took the aerobic granular sludge bioreactor(GSBR) and the nitrification sludge bioreactor(NSBR) where the most bacteria were selfnitrification bacteria as subjects. The inculation of E.coli K12(RP4) was conducted in different concentrations and frequencies in different reators. Culture counting and realtime quantitative PCR were used for counting the donor strain E.coli K12(RP4) and RP4 plasmid. Polymerase chain reaction-denaturing gradient gel eletrophoresis(PCRDGGE) was used to observe the diversity of AOB community structure. The main advantages of AOB were sequenced, and the sequencing results were compared and identified in the Gen Bank database. The qPCR was conducted to analyze changes in the number of AOB. The results would help to study the effect of ARGs on the ammonia removal efficiency, the flora of AOB and the mechanism in AOB metabolic activity. The results showed that the inculation of E.coli K12(RP4) in high concentration affected the removal efficiency of COD and NH4+-N. But the impact on NH4+-N was obvious, NH4+-N removal efficiency dropped to 32.8%, followed by 3 days were lower than 40%. From day 12 to day 17, it was still less than 70%. After day 18, it increased quickly to the level before inculation. AOB bacteria structure was stable, and the dominant AOB were Nitrosomonas and its high homology flora. The numbers of AOB in all size of sludge were stable before day 17, after day 18 quantities of AOB in flocculent sludge and smaller granular sludge increased. RP4 plasmid affected the metabolic activity of AOB, with the reduction of RP4 in GSBR, metabolic activity of AOB recovered gradually, newly-propagated AOB growed and increased, the NH4+-N removal efficiency was increased. In NSBR, the persistent inculation of E.coli K12(RP4) did not result in high concentration of donar strain in sludge, while the ratio of RP4 remained high consistently. RP4 plasmid transferred from heterotrophic bacteria to selfnitrification bacteria. The low concentration of E.coli K12(RP4) affected the rate of ammonia removal. The high concentration of E.coli K12(RP4) affected the ammonia removal efficiency, which reduced to 50%-70%, with ammonia and nitrite nitrogen being detected in effluent. The number and structure of AOB did not change in NSBR. Therefore, with the horizontal transfer of RP4 plasmid from heterotrophic bacteria to self-nitrification bacteria, the metabolic activities of AOB were affected and inhibited.