Effects Of Metabolic Uncouplers On Antibiotic Resistance Genes In Sludge Reduction Process

Antibiotic resistance genes (ARGs) as a new type of environmental pollutants, has raised scholars’ attention with its rapid emergence and spread in the environment. The sewage treatment plant is one of the major reservoirs of ARGs, a lot of studies have shown that high concentration of ARGs were detected in the sludge in sewage treatment plant. Also, the treatment and disposal of sludge has always been a thorny issue. Using uncoupling techniques for sludge reduction has been widely researched, and it’s effects on sludge reduction have also been recognized. While, as a new type of sludge reduction technology, the influences of metabolic uncouplers on ARGs which abundant in sludge have not yet been studied. In this paper, three kinds of metabolic uncouplers-3,3’,4’,5-tetrachlorosalicylanilide (TCS), p-nitrophenol (pNP), 2,4-dinitrophenol (DNP) were added to activated sludge, and the sludge yield was measured after short-term sludge culture, aslo twelve ARGs (tetC, tetG, tetO,tetW, tetX, sulⅠ, swl Ⅱ, ermB, ermF, dfrA1, dfrA12, blaTEM), as well as the class 1 integron integrase gene (intI1) and 16S rRNA gene were quantitatively detected after the reduction process. In addition, the study aslo used a multi-drug resistant plasmid RP4 which was charactered with conjugative transfer to initially investigate the mechanisms of the effects of metabolic uncouplers on ARGs at the microbial level. The results are as follows:1) The three metabolic uncouplers (TCS, pNP, DNP) showed a good reduction effect in activated sludge. For TCS, it reached the best reduction effect at the concentration of 4 mg/L, where the reduction rate of sludge is 94%; For pNP, it achieved the best reduction effect at the concentration of 15 mg/L, where the reduction rate of sludge is 79%; For DNP, it achieved the best reduction effect at the concentration of 10 mg/L, where the reduction rate of sludge is 65%. Besides, the addition of three metabolic uncouplers sludge had influence on the removal of COD, total nitrogen (TN) and total phosphorus (TP).2) TCS、pNP and DNP significantly promote the abundance of antibiotic resistance genes (ARGs) in sludge reduction process. And the concentration experiments showed that when the concentration of TCS. pNP and DNP was 4,15 and 10 mg/L respectively, the maximum relative abundance of ARGs is about 8.3,3.3 and 1.3 times of the control group. Among the three uncouplers, TCS greatly facilitated the dissemination of ARGs, DNP had least influence on ARGs. Within a certain range of concentrations, uncouplers played a significant role in promoting the proliferation of ARGs, when the concentration continued to increase, the toxicity of uncouplers will greatly inhibit the growth of bacteria or even cause death, thus leading to the inhibition of proliferation of ARGs, the abundance of ARGs decreased.3) After the sludge reduction with uncouplers, the abundance of five kinds of antibiotic resistance genes and the abundance of the mobile genetic element intI1 have shown a significant correlation (p<0.05), indicated that the change of the abundance of ARGs under the effect of uncouplers closely related to the horizontal gene transfer.4) TCS and pNP played a significant role in promoting the conjugative transfer of plasmid RP4 between Escherichia coli. And the transfer frequency increased with the mating time, peaked at 18 h, then decreased. When the concentration of TCS is 4 mg/L, the highest conjugation frequency of plasmid RP4 is about 24 times of the control group; when the concentration of pNP is 15 mg/L, the highest conjugation frequency of plasmid RP4 is about 6 times of the control group. Conjugation is an important way of horizontal gene transfer, indicating the mechanism of metabolic uncouplers increasing the abundance of ARGs is due to the role of horizontal gene transfer.