A meta-analysis reveals that operational parameters influence levels of antibiotic resistance genes during anaerobic digestion of animal manures.

Anaerobic digestion (AD) has shown the potential to reduce the numbers and types of antibiotic-resistance genes (ARG) present in animal manures. However, the variability of the results has limited the ability to draw solid conclusions. To address this issue, we performed a series of meta-analyses to evaluate how AD of pig, cattle, and dairy manures affects ARG levels and how different parameters, such as temperature, pH, digestion times, and the addition of other substances (e.g., solids, antibiotics) influence ARG changes. Twenty studies with enough details on changes in ARG levels during the AD process were identified and used for the meta-analyses. The results suggested that AD could significantly reduce ARG levels regardless of the conditions of the process. Also, thermophilic AD was more effective than mesophilic AD at reducing ARGs, although this difference was only significant for pig manures. The results also suggested that long digestion times (>50 days) yielded better ARG reduction rates, and that the addition of solids from an external source (co-digestion) negatively affected the efficiency of ARG reduction. In general, the results suggested that ARG changes during AD could be linked to the abundance and activity of hydrolytic communities.

Performance and recovery of nitrifying biofilm after exposure to prolonged starvation.

Achieving consistent ammonia removal in post-lagoon processes faces two major challenges impacting nitrifiers due to the unique seasonal variation of lagoon-based systems: summer to winter temperature drop and summer to fall ammonia starvation period while lagoon is removing ammonia. The objective of this study was to follow microbial diversity and define conditions that could overcome these challenges in a post-lagoon moving bed biofilm reactor (MBBR) operated at an initial surface area loading rate (SALR) of 0.3 g-NH4-N m-2d-1 from mesophilic (20 °C) to psychrophilic (4 °C). Initially the temperature was maintained at 20 °C and decreased to 10 °C until steady state was achieved. During starvation conditions (i.e., continuous, intermittent and no aeration without inflow; decanted media; and intermittent and continuous ammonia supplement) the temperature was decreased by 2 °C per week until 4 °C. The results indicated that operational procedures, such as intermittent ammonia supplement with SALR of 0.15 g-NH4-N m-2d-1 could improve performance with 80% ammonia removal achieved immediately after starvation period. Intermittent ammonia supplement had produced the greatest biofilm preservation comparable to the initial load with the highest specific and surface area removal rates. In the recovery phase (initial load restoration) 10 days were required to reestablish performance above 95% ammonia removal. When temperature was decreased from mesophilic to psychrophilic, the microbial diversity was found higher when starving biofilm compared to the control operated at the initial load while it converged to a similar population over recovery. The main actors associated to nitrification enriched at psychrophilic conditions were Proteobacteria and Bacteriodotes at phyla level. Ammonia oxidation to nitrite was mainly driven by the order Burkholderiales and nitrite oxidation to nitrate by Pseudomonadales. This procedure should be considered in the implementation of full-scale post-lagoon MBBR technologies to ensure reliable, robust, and consistent performance despite the inherent seasonal variability of lagoon-based processes.

Response of Microbial Community to Induced Failure of Anaerobic Digesters Through Overloading With Propionic Acid Followed by Process Recovery.

In order to effectively use microbial-based strategies to manage anaerobic digesters, it is necessary to distinguish between community shifts that are part of the natural dynamic of the system and shifts caused by environmental or operational disturbances. The objective of this research study was to evaluate the significance of changes in the microbial community of anaerobic digesters during failure in correlation to operational parameters such as an organic acid overload. Five continuously stirred 0.5 L reactors were set-up as semi-continuously-fed, mesophilic dairy manure digesters with a 30-day hydraulic retention time. After a 120-day stabilization period, two digesters were kept as controls, while the organic loading rates in the triplicate set were increased step-wise to ultimately provide a shock-load leading to failure using propionic acid spikes. Acidosis resulting in near cessation of biogas and termination of methane production occurred between 4 and 7 weeks, after which all the digesters continued to be fed only dairy manure. The shock loading of propionic acid led to an accumulation of mainly acetate and propionate, with low levels of iso-butyrate, butyrate, iso-valerate, and valerate. High-throughput Illumina sequencing of the V4 region of the bacterial and archaeal 16S rRNA gene in digester samples showed a significant change in the microbial community composition during propionic acid overload, followed by a return to the original composition with regular feedstock. Bacterial genera whose relative abundance decreased during the inhibition stage included Sedimentibacter, Syntrophomonas, TSCOR003.O20, and Marinilabiaceae, while the relative abundance of Lachnospiraceae, Ruminococcus, Mogibacteriaceae, Pyramidobacter, and Bacteroides increased. The relative abundance of dominant methanogens, Methanosarcina and Methanobacterium, although initially resistant, were decreased (from 91.71 to 12.14% and from 2.98 to 0.73%, respectively) during inhibition, while Methanobrevibacter and Methanosphaera that were prominent in the manure feedstock increased from 17.36 to 79.45% and from 0.14 to 1.12%, respectively. Shifts in bacterial and archaeal compositions, back to their pre-shock steady state after failure, highlight the digester's microbial resilience and recovery potential.

Effect of mesophilic anaerobic digestion on the resistome profile of dairy manure.

The effect of mesophilic anaerobic digestion (AD) on the resistome profile of manures from two different dairy farms was evaluated using a metagenomic approach. A total of 187 unique Antibiotic resistance genes (ARGs) for 17 different classes of antibiotics were detected in raw (undigested) manures. The results indicate that regardless of the origin of the dairy manure, mesophilic AD was capable of reducing or enriching the relative abundance of some ARGs. The main driver of these changes was strongly correlated with the evolution of the microbial community during the AD process. Putative ARG hosts were suggested by analyses of the co-occurrence of microbial groups and ARGs. Finally, network analyses revealed that mesophilic AD could also reduce the co-occurrence of different groups of ARGs potentially located in the same genetic elements. Our results provide valuable insights into the microbial mechanisms driving the diversity and abundance of ARGs during mesophilic AD.

Effect of ceftiofur on mesophilic anaerobic digestion of dairy manure and the reduction of the cephalosporin-resistance gene cmy-2.

The effect of ceftiofur (CEF), a commonly used antibiotics on dairy farms, on the performance and stability of mesophilic batch anaerobic digestion (AD) of dairy manure was evaluated in terms of methane production, organic matter removal (COD, dCOD, TS, and VS), and synthesis of end-products (VFAs, CO2, and H2). The results indicated that only CEF concentrations of 10 mg/L or higher significantly affected the performance of the AD process, although the overall stability was not compromised. Biochemical analyses suggested that hydrolytic microorganisms were the most affected by the presence of CEF leading to lower COD removal, whereas acetogens were only temporarily slowed down. Methanogens, on the other hand, were not directly affected by any of the CEF concentrations tested (0.2-250 mg/L). Additionally, the presence of CEF was shown to alter the incidence of the cephalosporin-resistance marker, cmy-2, although an overall reduction was achieved in 15-day batch anaerobic digestion trials.