Kinetic and microbial response of activated sludge community to acute and chronic exposure to tetracycline
Introduction
Tetracyclines, are among the most widely used antibiotics; They were discovered in the 1940’s, are broad-spectrum antibiotics that work against a large number of gram-negative and –positive bacteria, Tetracycline group of antibiotics are also strong chelating agents, which supports their antimicrobial properties [1]. They inhibit protein synthesis by hindering the binding of amiacyl-tRNA with the ribosome [1,2].
In activated sludge systems, tetracycline is removed mostly by sorption onto sludge [3,4]. This removal mechanism may be the result of tetracycline’s tendency to form very low solubility complexes by binding with divalent cations like calcium, magnesium, cadmium, cobalt and magnesium [5,6]; it may also relate to its strong chelating potential [1]. Alexy et al [6] studied the biodegradability characteristics of antibiotics by Closed Bottle Biodegradability Test (301D) [7], and obtained tetracycline removal up to 75% for 3.09 mg TET/L. Similarly, at initial tetracycline concentrations of 10, 20 and 30 mg/L, Shi et al [8] characterized the removal process by quick sorption and slow biodegradation of the compound.
Prescription of high doses of antibiotics by doctors and un-prescribed usage of these substances increases the inflow of antibiotics into natural habitats. Antibiotic substances, causing pollution in receiving waters are resistant to biodegradation and therefore they tend to persist in the environment, which increases the probability of environmental organisms to become resistant to these substances. Finally, today in most of the tested water bodies and soil samples antibiotic resistance genes are being detected [9,10], proving the effect of antibiotics in natural habitats. Besides the environmental concerns, increasing clinical resistance leads to inability of treating illnesses by taking antibiotics. Additionally, resistant bacteria in subterranean water bodies may reach surface waters, which are used as drinking water supplies, and may cause illnesses [11]. Therefore, antibiotic resistance constitutes a major problem for human and animal and therefore for World health [10].
Wastewater treatment plants are shown to act as reservoirs of human and animal bacteria, and antibiotic resistance genes are leaving these reservoirs through effluent reaching the receiving waters [[12], [13], [14]]. The activated sludge process, i.e the most widely used biological treatment system for wastewaters, are diverse and dynamic ecosystems and have large potential for exchange of genetic information [15]. Different studies showed, that activated sludge ecology contains high amounts and wide diversities of antibiotic resistance genes [12,14,16].
Resistance to tetracyclines can be explained by different mechanisms, such as efflux pumps, ribosomal protection proteins and enzymatic mechanisms. In the literature, 61 tet and otr genes have been defined coding resistance against tetracyclines, among which 34 are coding efflux pumps, while 13 are coding ribosomal protection proteins. In addition to these, there are 13 genes for enzymatic resistance and 1 gene for an unknown mechanism [17]. Moreover, tet genes that can be detected in gram-positive and gram-negative bacteria are also different [18]. However, the number of tet genes that can be found in water environments is lower; the tet genes found in activated sludge systems are even more limited.
In this context, the main aim of this study was to reflect the relationship between the kinetics of biochemical reactors and the community composition by determine the acute and chronic impact of tetracycline on the kinetic properties of the microbial biomass and its structure. Tetracycline resistance gene profile was also determined. Evaluation mainly relied on respirometry and model evaluation of the oxygen utilization rate (OUR) profiles generated at critical phases of the experimental period, together with microbial population analysis by 454-pyrosequencing, where the microbial community sustained in the reactor was continuously exposed to TET dosing. Additionally, sludge samples taken from an activated sludge system chronically fed with tetracycline were qualitatively analyzed for the presence of tet A, B, C, D, E, G, K, L, otrB and tet M, O genes, covering both efflux protein and ribosomal protection genes, respectively.
Section snippets
Reactor setup and operation
The seed sludge was taken from the aeration tank of a domestic wastewater treatment plant achieving nutrient removal. Using the seed sludge a 14 L laboratory-scale fill/draw reactor (θH = 1 day) was established and acclimated to peptone-meat extract mixture (peptone mixture) and operated until it reached steady state, at sludge age – sludge retention time (SRT) – of 10 days. Dissolved oxygen (DO) concentration in the reactor was kept above 3.0 mg/L at all times. Peptone mixture [19] was
Evaluation of oxygen uptake rate profiles
The OUR curves obtained different experimental runs are given in Fig. 1, where the effect of TET can even be seen naked eye, since the OUR profile changes with time of exposure.
In order to be able to determine the impact of tetracycline, the state with no impact of TET was assessed primarily. Therefore. Run 1 was taken as a baseline. OUR curve showing the biodegradation of peptone-meat extract mixture is given in Fig. 2. The profile makes a peak and continues to drop until the system reaches a
Conclusions
This study presented conclusive experimental evidence that continuous exposure to tetracycline caused complete suppression of substrate storage aside from mild inhibition on the growth kinetics and increase in endogenous decay rates by 1.5 fold. Increase in endogenous decay level was associated with maintenance energy dictated by presence and production of antibiotic resistance genes. Additionally, tetracycline exerted a significant binding action on available organic carbon, leading to less
Acknowledgements
This study was supported by Turkish Academy of Sciences as part of Fellowship Program for Integrated Doctoral Studies. Authors thank The Scientific Research Fund of Istanbul Technical University (ProjectNr:33680 and ProjectNr:33742) and The Scientific and Technological Research Council of Turkey. Authors thank University of Kiel Institute of Clinical Molecular Biology for their technical assistance.
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