24The study evaluates the chronic impact of the antibiotic tetracycline on the biodegradation of 25 organic substrate under anaerobic conditions. The experiments involved an anaerobic 26 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT2 sequencing batch reactor fed with a synthetic substrate mixture including glucose, starch and 1 volatile fatty acids, and operated in a sequence of different phases with gradually increasing 2 tetracycline doses of 1.65 -8.5 mg/L, for more than five months. Tetracycline exerted a 3 terminal/lethal effect at 8.5 mg/L on the microbial community under anaerobic conditions, 4 which caused the inhibition of substrate/COD utilization and biogas generation and leading to 5 a total collapse of the reactor. The microbial activity could not be recovered and re-started 6 within a period of more than 10 days, even after stopping tetracycline dosing. At lower doses, 7 substrate utilization was not affected but a reduction of 10-20% was observed in the 8 biogas/methane generation, suggesting that substrate utilization of tetracycline to the 9 biomass was limiting their bioavailability. During the experiments, tetracycline was partially 10 removed either through biodegradation or conversion into its by-products. The adverse long-11 term impact was quite variable for fermenting heterotrophic and methanogenic fractions of 12 the microbial community based on changes inflicted on the composition of remaining/residual 13 organic substrate. 14 15 16

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Cattle manure is frequently used as an inoculum for the start-up of agricultural biogas plants or as a co-substrate in the anaerobic digestion of lignocellulosic feedstock. Ruminal microbiota are considered to be effective plant fiber degraders, but the microbes contained in manure do not necessarily reflect the rumen microbiome. The aim of this study was to compare the microbial community composition of cow rumen and manure with respect to plant fiber-digesting microbes. Bacterial and methanogenic communities of rumen and manure samples were examined by 454 amplicon sequencing of bacterial 16S rRNA genes and mcrA genes, respectively. Rumen fluid samples were dominated by Prevotellaceae (29%), whereas Ruminococcaceae was the most abundant family in the manure samples (31%). Fibrobacteraceae (12%) and Bacteroidaceae (13%) were the second most abundant families in rumen fluid and manure, respectively. The high abundances of fiber-degrading bacteria belonging to Prevotellaceae and Fibrobacteraceae might explain the better performance of anaerobic digesters inoculated with rumen fluid. Members of the genus Methanobrevibacter were the predominant methanogens in the rumen fluid, whereas methanogenic communities of the manure samples were dominated by the candidate genus Methanoplasma. Our results suggest that inoculation or bioaugmentation with fiber-digesting rumen microbiota can enhance the anaerobic digestion of lignocellulosic biomass.

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The aim of this study was to investigate the effect of a common veterinary antibiotic in biogas plants. 20 mg/kg of oxytetracycline was intramuscularly injected into a cow and its concentration in manure, which was sampled daily during the following 20 days, was measured. A total of 20 % of the injected oxytetracycline was detected in manure. Collected manure samples on days 1, 2, 3, 5, 10, 15, and 20 were digested in triplicate serum bottles at 37 °C for 30 days. Control serum bottles produced 255 ± 13 mL biogas, whereas 50-60 % inhibitions were obtained for the serum bottles operated with samples collected for the 5 days after medication. Multivariate statistics used for the evaluation of FISH results showed that Methanomicrobiales were the main methanogenic group responsible for most of the biogas production. Numbers of active Bacteria and Methanomicrobiales were negatively correlated with the presence of oxytetracycline, whereas Methanosarcinales and Methanobacteriales were less affected.