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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Universidade do Minho: RepositoriUM Chemosphere 117 (2014) 295–302 Contents lists available at ScienceDirect Chemosphere journal homepage: www.elsevier.com/locate/chemosphere Influence of tetracycline on the microbial community composition and activity of nitrifying biofilms ⇑ Maria Matos a, , Maria A. Pereira a, Pier Parpot b, António G. Brito a,d, Regina Nogueira c a CEB – Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal b Centre of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal c ISAH – Institute of Sanitary Engineering and Waste Management, University of Hannover, Welfengarten 1, D-30167 Hannover, Germany d Institute of Agronomy, Department of Biosystems Sciences and Engineering, University of Lisbon, Tapada da ajuda, 1349-017 Lisboa, Portugal highlights Tetracycline did not affect the removal of carbon and nitrogen. The antibiotic affected the bacterial composition of the biofilms. The tetracycline removal was poor (28%). Biodegradation was probably the main removal mechanism of the antibiotic. The occurrence of tet(S) was influenced by the presence of tetracycline. article info abstract Article history: The present work aims to evaluate the bacterial composition and activity (carbon and nitrogen removal) Received 14 February 2014 of nitrifying biofilms exposed to 50 lgLÀ1 of tetracycline. The tetracycline removal efficiency and the Received in revised form 27 June 2014 occurrence of tetracycline resistance (tet) genes were also studied. Two sequencing batch biofilm reactors Accepted 28 June 2014 (SBBRs) fed with synthetic wastewater were operated without (SBBR1) and with (SBBR2) the antibiotic. Both SBBRs showed similar organic matter biodegradation and nitrification activity. Tetracycline removal Handling Editor: O. Hao was about 28% and biodegradation was probably the principal removal mechanism of the antibiotic. Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of the bacterial community Keywords: showed shifts leading to not only the fading of some ribotypes, but also the emergence of new ones in Antibiotics the biofilm with tetracycline. The study of the tet genes showed that tet(S) was only detected in the Bacterial community biofilm with tetracycline, suggesting a relationship between its occurrence and the presence of the Nitrifying biofilms antibiotic. Tetracycline removal Ó 2014 Elsevier Ltd. All rights reserved. Resistance genes 1. Introduction 2013). Due to all these facts, antibiotics are emerging as a new group of pollutants in wastewater and a significant number of Antibiotics have been widely used therapeutically in human studies focused on their presence and elimination in WWTPs have and veterinary medicine as well as non-therapeutically in animal been reported in the literature as reviewed by Miège et al. (2009) production and many of them are excreted unchanged as active and Le-Minh et al. (2010). According to them, the main mecha- compounds into wastewater. The presence of antibiotics in waste- nisms involved in the removal of antibiotics from wastewater are water and their persistence through wastewater treatment plants biodegradation, sorption onto extracellular polymeric substances (WWTPs) potentially contribute to their release into surface (EPS), filtration and chemical oxidation. However, since WWTPs waters, where they can accumulate and reach detectable and rely on the composition and activity of their microbial communi- biologically active concentrations. Dissemination of antibiotics in ties, it is also important to study if antibiotics in the wastewater the environment can lead to the emergence and spread of antibiot- might negatively affect the diversity and activity of the microbial ics resistance, which can reduce their therapeutic potential against communities in biological systems. pathogens, thus increasing risks to public health (Rizzo et al., Biodegradation of organic matter by heterotrophic bacteria and biological oxidation of ammonia via nitrite to nitrate by autotroph- ⇑ Corresponding author. Tel.: +351 253 604414; fax: +351 253 678 986. ic bacteria, a process known as nitrification, are key processes of E-mail address: [email protected] (M. Matos). the biological wastewater treatment. Typical heterotrophic http://dx.doi.org/10.1016/j.chemosphere.2014.06.094 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved. 296 M. Matos et al. / Chemosphere 117 (2014) 295–302 bacteria in WWTPs are affiliated to the alpha, beta and gamma to complete the 10% of the reactors VT. During the aerated phase, subclasses of the Proteobacteria (Seviour and Nielsen, 2010). Other an airflow of 2 L minÀ1 was supplied through membrane diffusers. members affiliated to the Bacteroidetes, Firmicutes and Actinobac- The applied airflow ensured that biofilm carriers were moving teria phyla have also been frequently retrieved (Seviour and freely along with the liquid in the reactors. The composition of Nielsen, 2010). Autotrophic nitrifying bacteria in WWTPs encom- the synthetic wastewater simulated a municipal wastewater pass the ammonia oxidizing bacteria (AOB) that are almost exclu- (Nopens et al., 2001). SBBR1 was operated without tetracycline sively restricted to the Betaproteobacteria and the nitrite oxidizing (control), while in SBBR2 the synthetic wastewater was supple- bacteria (NOB) that are distributed among the Alphaproteobacte- mented with the antibiotic (50 lgLÀ1). The reactors were operated ria, in particular within the Nitrobacter genus and the phylum for 60 d. Regular cleaning of feed storage vessels, feed lines and Nitrospirae, assigned to the Nitrospira genus (Daims and Wagner, reactors was performed to avoid the proliferation of microorgan- 2010). There are a limited number of studies that report the effect isms on tubes and walls. of antibiotics in the community composition and activity of hetero- trophs and nitrifiers in WWTPs. Collado et al. (2013) reported bac- 2.2. Analytical methods terial community shifts in a lab-scale sequencing batch reactor (SBR) fed with synthetic wastewater containing 50 lgLÀ1 of sulfa- Total kjeldahl nitrogen (TKN), N–NH+, N–NOÀ, N–NOÀ, total methoxazole, but they did not observe any impact in the removal 4 2 3 chemical oxygen demand (COD) and total suspended solids (TSS) of carbon and nitrogen as well as in the number of resistance were determined according to the Standard Method (APHA, genes. Deng et al. (2012) showed that the performance of a 2005). Samples for N–NH+, N–NOÀ and N–NOÀ analyses were full-scale biosystem fed with wastewater containing high concen- 4 2 3 immediately filtered through a 0.45 lm pore size filter (Advantec, trations of antibiotics (mainly streptomycin at 3.96 mg LÀ1) was Dublin, CA, USA). Biofilm concentration (mg carrierÀ1) was esti- maintained due to the adjustment of the bacterial, archaeal and mated according to Matos et al. (2012). Tetracycline was deter- eukaryal communities. Furthermore, they suggested that the mined by solid phase extraction (SPE) followed by high increase of Betaproteobacteria and Bacteroides observed in the performance liquid chromatography–mass spectrometry (HPLC– aerobic reactor of the biosystem was related with their ability to MS). Before SPE extraction, samples were filtered through a acquire antibiotic resistance genes. In contrast, Wunder et al. 0.45 lm pore size filter. Then, the pH of the sample was adjusted (2013) reported that ciprofloxacin (3.33 lgLÀ1) can affect both to <3 with 40% H SO and 50 lLof5%NaEDTA as well as 50 lL the structure and activity of biofilms growing in a continuous-flow 2 4 2 of 50 lgmLÀ1 chlorotetracycline solution (internal standard) were rotating annular bioreactor, while erythromycin and sulfamethox- added to the sample. The SPE procedure consisted of the precondi- azole (at similar concentration as ciprofloxacin) did not affect the tion of an Oasis HLB cartridge (30 mg mLÀ1, Waters, Miliford, MA, environmental bacterial biofilms. Wunder and his co-workers also USA) with 1 mL of methanol, 1 mL of 0.5 N HCl and 1 mL of deion- found that a mixture of the three antibiotics at 0.33 lgLÀ1 (for ized water sequentially. Then, 5 mL of sample was extracted with each compound) is unlikely to affect the performance of the bio- the HLB cartridge at a flow rate of approximately 5 mL minÀ1 and film process, at least in terms of degradation of easily biodegrad- the cartridge was washed with 1 mL of 5% methanol aqueous solu- able substrates. tion. Finally, the antibiotic was eluted with 1 mL of methanol. The The main goal of the present work was to evaluate the potential extract was dried under a gentle stream of N and the dried residue influence of tetracycline in the composition and activity of the 2 was dissolved with 1 mL of mobile phase B (0.1% acid formic in microbial community (total and nitrifying bacteria) in biofilm reac- acetonitrile). The HPLC system consisted of a HPLC Pump (Finnigan tors. The removal of tetracycline, the sorption of tetracycline onto Surveyor Plus), an autosampler (Finnigan) and a photodiode array EPS as well as the occurrence of tetracycline resistance (tet) genes detector (Finnigan). Tetracycline and the internal standard were were also studied. Tetracycline was the selected antibiotic because separated using a Synergi Hydro-RP column (150 mm Á 4.6 mm) it is commonly detected in wastewater (Miège et al., 2009). For packed with 4 lm size particles (Phenomenex, Torrance, CA,
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  • Effects of Cultivation Ages and Modes on Microbial Diversity in the Rhizosphere Soil of Panax Ginseng

    Effects of Cultivation Ages and Modes on Microbial Diversity in the Rhizosphere Soil of Panax Ginseng

    J Ginseng Res 40 (2016) 28e37 Contents lists available at ScienceDirect Journal of Ginseng Research journal homepage: http://www.ginsengres.org Research article Effects of cultivation ages and modes on microbial diversity in the rhizosphere soil of Panax ginseng Chunping Xiao, Limin Yang*, Lianxue Zhang, Cuijing Liu, Mei Han Cultivation Base of State Key Laboratory for Ecological Restoration and Ecosystem Management of Jilin Province and Ministry of Science and Technology, College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun, China article info abstract Article history: Background: Panax ginseng cannot be cultivated on the same land consecutively for an extended period, Received 12 February 2015 and the underlying mechanism regarding microorganisms is still being explored. Received in Revised form Methods: Polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE) and BIO- 27 March 2015 LOG methods were used to evaluate the microbial genetic and functional diversity associated with the Accepted 10 April 2015 P. ginseng rhizosphere soil in various cultivation ages and modes. Available online 30 April 2015 Results: The analysis of microbial diversity using PCR-DGGE showed that microbial communities were significantly variable in composition, of which six bacterial phyla and seven fungal classes were detected in Keywords: DGGE P. ginseng soil. Among them, Proteobacteria and Hypocreales dominated. Fusarium oxysporum, a soilborne discontinuous cultivation pathogen, was found in all P. ginseng soil samples except R0. The results from functional diversity suggested metabolic function that the microbial metabolic diversity of fallow soil abandoned in 2003 was the maximum and transplanted microbial composition soil was higher than direct-seeding soil and the forest soil uncultivated P.