Inhibition of Methane Fermentation by Antibiotics Introduced to Municipal Anaerobic Sludge †

Annually, a few thousand tons of antibiotics and their transformation products (metabolites and degradation products) are introduced to wastewater treatment plants (WWTPs) as a result of human and animal excretion, or dispose of expired or unused medications. Antibiotics present in wastes might inhibit their treatment processes for instance during methane fermentation. In this study, β-lactams, tetracycline’s, fluoroquinolones, sulphonamides and metronidazole were selected as inhibitors of methane fermentation of sewage sludge collected from municipal WWTP. The experiments were performed in two series with different concentrations of antibiotics. The biogas production did not significantly differ between series, and was from 151.7 ± 18.9 mL/g VS (in the bioreactor with metronidazole addition—II series) to 208.3 ± 11.9 mL/g VS (in the bioreactor with amoxicillin addition—I series). In the control sample biogas production was 203.7 ± 21.1 mL/g VS. The methane content in all experiments was from 61.3 ± 2.1% to 66.4 ± 3.1%. The results indicated that microorganisms in anaerobic sludge from municipal wastewater are highly resistant to antibiotics in the tested concentrations. Antibiotic present in wastewater probably caused of antibiotic resistance in bacteria.


Introduction
Biogas produced by methane fermentation of renewable feedstocks is considered as one of the alternatives to fossil fuels.In this energy recovery process organic compounds are oxidized and inorganic compounds are used as terminal electron acceptors.Generally, methane fermentation is divided into the stages, and each stage is conducted by different groups of microorganisms, forming an ecosystem of different trophic levels in the food chain.Four main stages are: hydrolysis of macromolecular compounds, acidogenesis (decomposition of hydrolyzed compounds to organic acids), acetogenesis (decomposition of organic acids to acetic acid) and methanogenesis.Depending on the stage, facultative and obligatory anaerobic microorganisms are involved in those processes.Hydrolysis is process of depolymerisation of complex organic compounds into monomers or dimers that are directly assimilated by bacteria.In the next stage-cidogenesis, the hydrolyzed products are decomposed into low molecular organic compounds.Essential products of further transformations are methane and carbon dioxide [1].
Antibiotics and their transformation products are dominant micropollutants in sludge because of specific properties like low degradation during wastewater treatment and high sorption to sludge.Annually, a few thousand tons of antibiotics and their transformation products (metabolites and degradation products) are introduced to wastewater treatment plants as a result of human and animal excretion, or dispose of expired or unused medications.The presence of these compounds in wastes is of a great interest due to their potential to cause negative effects-accumulation and spread of antibiotic resistance.Antibiotics present in wastes can inhibit the processes of their treatment for instance during methane fermentation.Gao et al. [2] confirmed the presence of fluoroquinolones, sulphonamides and macrolides in sewage sludge samples from 8 wastewater treatment plants.The presence of antibiotics can cause of antibiotic resistance in bacteria.
The aim of this study is to investigate the methane fermentation of sewage sludge collected from municipal wastewater treatment plant, with addition of antibiotics.In this study, β-lactams and tetracycline's were selected as inhibitors of methane fermentation, due to the fact that they account for approximately 95% of therapeutic arsenal proposed by pharmaceutical companies and are most frequently used in treatment of humans.Furthermore, metronidazole which is one of the mainstay drugs for the treatment of anaerobic infections as well as fluoroquinolones and sulphonamides which effect on Archaea, were chosen to investigate of inhibition of methane fermentation.

Materials and Methods
The experiments were performed in two series with different concentrations of antibiotics.The dose of 16 mg/kg of β-lactams (amoxicillin, cefuroxime disodium salt) and 8 mg/kg of tetracycline's (oxytetracycline, doxycycline, tigecycline) sulphonamides (sulfamethoxydiazine) and metronidazole and 2 mg/kg of fluoroquinolones (ciprofloxacin, nalidixic acid) were used.In the I series, the concentration of antibiotic was calculated based on the mass of introduced substrate (25 g).In the II series, the concentration of antibiotic was calculated based on the mass of inoculum and substrate (200 g).The substrate was sewage sludge from municipal WWTP in Olsztyn, Poland.The inoculum was anaerobic sludge from fermentation tank located in municipal WWTP in Olsztyn, Poland.The study was carried out in the methane potential analysis tool (AMPTS II Bioprocess Control).This device was used to measure the quantity of biogas produced.The quality of biogas was measured with an LXI 430 analyzer (GasData) and by using a gas chromatograph connected with thermal conductivity detector (GC-TCD) (Agillent 7890 A).Reactors of the volume of 500 mL were connected to the multifunctional agitation system.Mixing in the reactor run for 30 s each 10 min.Rotating speed was 100 rpm.Anaerobic conditions were achieved by continuous flushing of pure nitrogen through the sludge.Methane fermentation was carried out under mesophilic conditions at 37 °C for 7 days.The experiments were performed in duplicates.In the samples before and after fermentation the pH, the FOS/TAC ratio, the total solids (TS) and volatile solids (VS), and the nitrogen and phosphorus content were determined.The FOS/TAC ratio was determined with TitraLab AT1000 Series Titrator (Hatch).The TAC value is an estimation of the buffer capacity of the sample, and the FOS value indicates the volatile fatty acids content.The biomass samples were tested for the content of TS and VS with a gravimetric method.The concentration of TN and TP after mineralization of the samples was measured with HachLange cuvette tests.After testing for homogeneity of variance with Levene's test, the significance of differences between variants was tested with Tukey's HSD test.Differences were considered significant at p < 0.05.

Results and Discussion
In the I series, the biogas production was from 159.1 ± 10.8 mL/g VS (in the bioreactor with metronidazole addition) to 208.3 ± 11.9 mL/g VS (in the bioreactor with amoxicillin addition) (Figure 1).In the II series, the biogas production was from 151.7 ± 18.9 mL/g VS (in the bioreactor with metronidazole addition) to 198.3 ± 14.6 mL/g VS (in the bioreactor with nalidixic acid addition).In the control sample biogas production was 203.7 ± 21.1 mL/g VS.The concentration of antibiotics applied to bioreactors with methane fermentation did not significantly influence on biogas production.The methane content in all experiments was from 61.3 ± 2.1% to 66.4 ± 3.1%.Mean FOS/TAC ratio in the I series was 0.26 and in the II series 0.30 (Table 1).The FOS/TAC ratio indicates stable reactor performance.Higher values of this indicator in the II series, suggested inhibition of the last phase of methane production and accumulation of volatile fatty acids during fermentation.However, these results were not statistically significant.
In both series of the study, the lowest concentration of nitrogen was noted after fermentation of sewage sludge with oxytetracycline addition.However, the highest concentration of nitrogen were noted after methane fermentation of sewage sludge with nalidixic acid (series I) and with doxycycline addition (series II).Mean concentration of phosphorus after fermentation in the series I (6.8 mg/g) and in the series II (7.0 mg/g) was lower than in the control sample.The highest pH was noted in the control sample.The values of pH were similar in all the samples.The indicators of methane fermentation did not also significantly differ between the control bioreactor and bioreactors with antibiotics addition.
Table 1.The FOS/TAC ratio, concentration of nitrogen and phosphorus, and pH in digestate after biogas production from sewage sludge with antibiotics addition.Abbreviations: MET-metronidazole, AMO-amoxicillin, NA-nalidixic acid, DOXY-doxycycline, OXY-oxytetracycline, CEF-cefuroxime disodium salt, TIGE-tigecycline, SMX-sulfamethoxydiazine, CIP-ciprofloxacin, CONT-control; f.m.-fresh mass.No differences between samples suggest that antibiotics did not inhibit methane fermentation.The presence of antibiotics in wastewater might cause of antibiotic resistance in bacteria.Antibiotic resistance is determined by genes located on the bacterial chromosome or mobile elements, such as plasmids, transposons and integrons [3,4].These antibiotic resistance genes can spread easily in closely related bacteria and even in bacteria from various species or genera.Wastewater treatment plants are hot spots for gene transfer [5][6][7] due to the availability of nutrients, supportive temperature, high density of microbial communities, presence of donors and recipients, as well as factors that contribute to selective pressure [8].The results indicated that microorganisms in anaerobic sludge from municipal wastewater are highly resistant to antibiotics in the tested concentrations.