Behaviour of oxytetracycline in two-phase anaerobic cattle manure digesters and its effects on microbial communities

Abstract

Antibiotics used in veterinary practice may have inhibitory effects on microorganisms in anaerobic digestion of manure for biogas production as a renewable energy. Since microbial community plays a key role in the digestion process, defining the effects of antibiotics on microbial diversity and activity can lead to improvements in better understanding of interactions between microbial communities and biochemical processes, thus achieving a more efficient system performance in terms of higher digestion capacity and biogas production. In this study, inhibitory effects and behaviour of commonly used veterinary antibiotic, oxytetracycline (OTC) on the system performance and biogas production in a two-phase anaerobic digestion of cattle manure was investigated, as well as degree of acidification of cattle manure was improved. In the first part, different combinations of batch anaerobic digesters were set-up to investigate the optimum operational conditions for acidogenic phase. The digesters were operated at different pH ranges between 5.0 and 6.0; and at different % total volatile solids (TVS). pH of 5.5±0.1 and 6% TVS were found to be optimal due to higher production of VFA and acidification rate. In the second part, a commercially available hydrolytic enzyme mixture was added in order to improve the acidification rate of the cattle manure in the acidogenic phase of a two-phase anaerobic digestion system. Better solubilization resulted in higher VFA productions which caused higher biogas productions than previously achieved. In the third part, 2 seperate two-phase anaerobic cattle manure digesters, one operated with non-medicated manure and the other operated with medicated manure, and furthermore, 2 single-phase digesters were operated for comparison in semi-continuous mode for 60 days. SRT/HRT for two-phase and single-phase operations were 5 days for acidogenic digesters, 15 days for methanogenic digesters and 20 days for single-phase digesters. Organic loading rates were 6.25±0.15 kg TVS/m3 day in two-phase digesters and 1.50±0.02 kg TVS/m3 day in single-phase digesters. Biogas yields of non-medicated two-phase and single-phase manure digesters were almost the same, 299±26 and 289±25 L biogas/kg-TVS added, respectively; however, non-medicated two-phase digester had higher methane yield as 173±15 L CH4/kg-TVS added, which was 150±13 L CH4/kg-TVS added in single-phase digester. In medicated manure digesters, OTC concentration of 3.11±0.12 mg/L caused 35% inhibition on biogas yield and 43% inhibition on methane yield in the two-phase digester. In single-phase digester, OTC concentration of 3.07±0.14 mg/L caused 47% and 52% inhibitions on biogas and methane production, respectively. Reduction in OTC concentrations could have been attributed to abiotic and biotic reduction mechanisms such as photodegradation, temperature, humidity, pH, dilution during feeding of digesters and other environmental conditions, binding to organics and microbial degradation. The results indicated that the two-phase anaerobic digestion performed more efficient in the presence of OTC with respect to the biogas and methane production. According to DGGE and FISH results, almost all bacterial and Archaeal species were negatively affected by OTC. During the digestion period, DGGE results showed that the bacterial diversity in acidogenic digesters and Archaeal diversity in methanogenic digesters were higher than single-phase digesters. Bacteria groups of Firmicutes and Gammaproteobacteria; and Archaeal groups of Methanobacteriales and Methanosarcinales were found as the most abundant microorganisms. FISH results indicated that the relation between digestion period and the activity of microorganisms were mostly effective on Firmicutes and Gammaproteobacteria, showing a decreasing trend towards the end of digestion. In methanogenic activity, OTC presence was comparatively more effective on Methanosarcinales. The activity of Methanobacteriales and Methanosarcinales showed a more stable trend compared to bacteria; however, the ratios of active methanogenic cells were higher in methanogenic digesters compared to single-phase digesters, both in the absence and presence of OTC.