Bahar Ince

Chronic impact of tetracycline on the biodegradation of an organic substrate mixture under anaerobic conditions

The study evaluates the chronic impact of the antibiotic tetracycline on the biodegradation of organic substrate under anaerobic conditions. The experiments involved an anaerobic sequencing batch reactor fed with a synthetic substrate mixture including glucose, starch and volatile fatty acids, and operated in a sequence of different phases with gradually increasing tetracycline doses of 1.65-8.5 mg/L, for more than five months. Tetracycline exerted a terminal/lethal effect at 8.5 mg/L on the microbial community under anaerobic conditions, which caused the inhibition of substrate/COD utilization and biogas generation and leading to a total collapse of the reactor. The microbial activity could not be recovered and re-started within a period of more than 10 days, even after stopping tetracycline dosing. At lower doses, substrate utilization was not affected but a reduction of 10-20% was observed in the biogas/methane generation, suggesting that substrate utilization of tetracycline to the biomass was limiting their bioavailability. During the experiments, tetracycline was partially removed either through biodegradation or conversion into its by-products. The adverse long-term impact was quite variable for fermenting heterotrophic and methanogenic fractions of the microbial community based on changes inflicted on the composition of remaining/residual organic substrate.

Application of real-time PCR to determination of combined effect of antibiotics on Bacteria, Methanogenic Archaea, Archaea in anaerobic sequencing batch reactors.

This study evaluated the long-term effects of erythromycin-tetracycline-sulfamethoxazole (ETS) and sulfamethoxazole-tetracycline (ST) antibiotic combinations on the microbial community and examined the ways in which these antimicrobials impact the performance of anaerobic reactors. Quantitative real-time PCR was used to determine the effect that different antibiotic combinations had on the total and active Bacteria, Archae and Methanogenic Archae. Three primer sets that targeted metabolic genes encoding formylterahydrofolate synthetase, methyl-coenzyme M reductase and acetyl-coA synthetase were also used to determine the inhibition level on the mRNA expression of the homoacetogens, methanogens and specifically acetoclastic methanogens, respectively. These microorganisms play a vital role in the anaerobic degradation of organic waste and targeting these gene expressions offers operators or someone at a treatment plant the potential to control and the improve the anaerobic system. The results of the investigation revealed that acetogens have a competitive advantage over Archaea in the presence of ETS and ST combinations. Although the efficiency with which methane production takes place and the quantification of microbial populations in both the ETS and ST reactors decreased as antibiotic concentrations increased, the ETS batch reactor performed better than the ST batch reactor. According to the expression of genes results, the syntrophic interaction of acetogens and methanogens is critical to the performance of the ETS and ST reactors. Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors.

Development of antibiotic resistance genes in microbial communities during long-term operation of anaerobic reactors in the treatment of pharmaceutical wastewater.

Biological treatment processes offer the ideal conditions in which a high diversity of microorganisms can grow and develop. The wastewater produced during these processes is contaminated with antibiotics and, as such, they provide the ideal setting for the acquisition and proliferation of antibiotic resistance genes (ARGs). This research investigated the occurrence and variation in the ARGs found during the one-year operation of the anaerobic sequencing batch reactors (SBRs) used to treat pharmaceutical wastewater that contained combinations of sulfamethoxazole-tetracycline-erythromycin (STE) and sulfamethoxazole-tetracycline (ST). The existence of eighteen ARGs encoding resistance to sulfamethoxazole (sul1, sul2, sul3), erythromycin (ermA, ermF, ermB, msrA, ereA), tetracycline (tetA, tetB, tetC, tetD, tetE, tetM, tetS, tetQ, tetW, tetX) and class Ι integron gene (intΙ 1) in the STE and ST reactors was investigated by quantitative real-time PCR. Due to the limited availability of primers to detect ARGs, Illumina sequencing was also performed on the sludge and effluent of the STE and ST reactors. Although there was good reactor performance in the SBRs, which corresponds to min 80% COD removal efficiency, tetA, tetB, sul1, sul2 and ermB genes were among those ARGs detected in the effluent from STE and ST reactors. A comparison of the ARGs acquired from the STE and ST reactors revealed that the effluent from the STE reactor had a higher number of ARGs than that from the ST reactor; this could be due to the synergistic effects of erythromycin. According to the expression of genes results, microorganisms achieve tetracycline and erythromycin resistance through a combination of three mechanisms: efflux pumping protein, modification of the antibiotic target and modifying enzymes. There was also a significant association between the presence of the class 1 integron and sulfamethoxazole resistance genes.

Inhibition of Volatile Fatty Acid Production in Granular Sludge from a UASB Reactor

Abstract Inhibition of volatile fatty acids (VFA), namely acetate, butyrate, and propionate, on the activity of acetoclastic methanogens within a full-scale upflow anaerobic sludge blanket (UASB) reactor was investigated using specific methanogenic activity (SMA) test. SMA tests were carried out at acetate concentrations in a range of 1000–25,000 mg l−1, butyrate concentrations in a range of 3000–25,000 mg l−1 and propionate concentrations between 500–10,000 mg l−1. Maximum potential methane production (PMP) rates were obtained as 389 ml CH4 gTVS−1 .d−1 at 3000 mg l−1 acetate concentration, 432 ml CH4gTVS−1.d−1 at butyrate concentration of 5000 mg l−1, and 162 mlCH4gTVS−1.d−1 at 1000 mg l−1 propionate concentration. App. 50% and 100% inhibition occurred at acetate concentrations of 13,000 mg l−1 and 25,000 mg l−1, butyrate concentrations of 15,000 mg l−1 and 25,000 mg l−1, and propionate concentrations of 3500 mg l−1 and 5000 mg l−1, respectively.

Biogeographical distribution and diversity of bacterial and archaeal communities within highly polluted anoxic marine sediments from the marmara sea.

Physicochemical and microbiological characterization of anoxic sediments taken from seven highly polluted sites of the Marmara Sea was carried out. The 16S rRNA based microbial community structure analyses were performed using domain-specific PCR followed by denaturant gradient gel electrophoresis (DGGE) and sequencing of characteristic bands. The results showed that the microbial communities in these sediments were diverse and evenly distributed. Relating the prokaryotic and geochemical variables through statistical tools revealed that the microbial diversity in the sediments significantly related to depth, and S, Mn and Fe content of the sediments. Fermentative bacteria, denitrifying bacteria and hydrogenotrophic methanogens were dominant whereas sulfate reducing bacteria were absent in the DGGE patterns. This unusual microbial community structure implied that the newly discovered anaerobic methane oxidation coupled to denitrification process may occur in these subseafloor environments.

Use of PCR-DGGE based molecular methods to assessment of microbial diversity during anaerobic treatment of antibiotic combinations

As it is currently often not know how anaerobic bioreactors, e.g. for biogas production, react if the substrate is contaminated by toxic compounds like antibiotics. This study evaluated how anaerobic sequencing batch reactors were affected by amendments of different antibiotics and stepwise increasing concentrations. The compositions of microbial community were determined in the seed sludge using 16S rRNA gene clone libraries and PCR-DGGE analyses were used for the detection of microbial community changes upon antibiotics additions. According to PCR-DGGE results, the syntrophic interaction of acetogens and methanogens is critical to the performance of the reactors. Failure to maintain the stability of these microorganisms resulted in a decrease in the performance and stability of the anaerobic reactors. Assessment of DGGE data is also useful for suggesting the potential to control ultimate microbial community structure, especially derived from Gram-negative bacteria, through bioaugmentation to successful for antibiotic biodegradation.

Acute inhibitory impact of antimicrobials on acetoclastic methanogenic activity

The study evaluated the short-term inhibition impact of three antimicrobials, sulfamethoxazole, erythromycin and tetracycline, on the methanogenic activity of acclimated biomass fed with acetate. Batch reactors were inoculated each with a different antimicrobial concentration in the range of 1-1000 mg/L and they were operated during 6 days. Organic substrate removal was monitored by both soluble COD and acetate measurements, together with daily measurements of biogas and methane generation. While acetate was almost fully removed in all experiments, methane generation exhibited a significant drop with increasing antimicrobial doses. Almost complete methane inhibition was observed for antimicrobial doses above 500 mg/L. Together with adverse impact on process kinetics in the early phases of the experiments, the final acute impact of antimicrobials was on process stoichiometry, preventing complete utilization of acetate removed in metabolic reactions. The observed effect was found compatible with uncompetitive inhibition, which similarly exerts a binding impact on substrate-enzyme complex.

Assessment of anaerobic bacterial diversity and its effects on anaerobic system stability and the occurrence of antibiotic resistance genes

This study evaluated the link between anaerobic bacterial diversity and, the biodegradation of antibiotic combinations and assessed how amending antibiotic combination and increasing concentration of antibiotics in a stepwise fashion influences the development of resistance genes in anaerobic reactors. The biodegradation, sorption and occurrence of the known antibiotic resistance genes (ARGs) of erythromycin and tetracycline were investigated using the processes of UV-HPLC and qPCR analysis respectively. Ion Torrent sequencing was used to detect microbial community changes in response to the addition of antibiotics. The overall results indicated that changes in the structure of a microbial community lead to changes in biodegradation capacity, sorption of antibiotics combinations and occurrence of ARGs. The enhanced biodegradation efficiency appeared to generate variations in the structure of the bacterial community. The results suggested that controlling the ultimate Gram-negative bacterial community, especially Acinetobacter-related populations, may promote the successful biodegradation of antibiotic combinations and reduce the occurrence of ARGs.

Combined effect of erythromycin, tetracycline and sulfamethoxazole on performance of anaerobic sequencing batch reactors.

The combined effects of erythromycin-tetracycline-sulfamethoxazole (ETS) and sulfamethoxazole-tetracycline (ST) antibiotics on the performance of anaerobic sequencing batch reactors were studied. A control reactor was fed with wastewater that was free of antibiotics, while two additional reactors were fed with ETS and ST. The way in which the ETS and ST mixtures impact COD removal, VFA production, antibiotic degradation, biogas production and composition were investigated. The effects of the ETS mixtures were different from the ST mixtures, erythromycin can have an antagonistic effect on sulfamethoxazole and tetracycline. The anaerobic pre-treatment of these antibiotics can represent a suitable alternative to the use of chemical treatments for concentrations at 10 mg/L of S and 1 mg/L of T; 2 mg/L of E, 2 mg/L of T and 20 mg/L of S for the ST and ETS reactors respectively, which corresponds to min 70% COD removal efficiency.

Assessment of Biogas use as an Energy Source from Anaerobic Digestion of Brewery Wastewater

Energy recovery from a crossflow ultrafiltration (UF) membraneunit employed in order to improve the performance of an anaerobic contact digester for the treatment of brewery wastewater was assessed. The performance of the pilot-scale anaerobic UF membrane system was studied for over 15 months. At steady-state conditions, an organic loading rate of 28.5 kg COD m-3 d-1, a hydraulic retention time of 4.2 days and overall COD and BOD removal efficiencies of 99% and almost 100% were achieved, respectively. Percent methane in biogas was found to be in a range of 67–79% with the corresponding methane yield of 0.28–0.35 m3 CH4 kg-1 CODremoved. The potential energy recovery from the system treating brewery wastewater at an OLR of 28.5 kg COD m-3 d-1 was 87 MJ d-1 which would enable to maintain all energy requirements of the feed pump, mixing and heating of the reactor contents. In addition to this, 71% of the energy requirement for recirculating the reactor content through the membranes would also be recovered.