Organic loading rate (OLR) is crucial for determining the stability of dry anaerobic digestion (AD). Digestate recirculation contributes to reactor stability and enhances methane production. Nevertheless, the understanding of how OLR and digestate recirculation affect the abundance and diversity of antibiotics and antibiotic resistance genes (ARGs), as well as the mechanisms involved in the dissemination of ARGs, remains limited. This study thoroughly investigated this critical issue through a long-term pilot-scale experiment. The metabolome analyses revealed the enrichment of various antibiotics, such as aminoglycoside, tetracycline, and macrolide, under low OLR conditions (OLR ≤ 4.0 g·VS/L·d) and the reactor instability. Antibiotics abundance decreased by approximately 19.66-31.69 % during high OLR operation (OLR ≥ 6.0 g·VS/L·d) with digestate recirculation. The metagenome analyses demonstrated that although low OLR promoted reactor stability, it facilitated the proliferation of antibiotic-resistant bacteria, such as Pseudomonas, and triggered functional profiles related to ATP generation, oxidative stress response, EPS secretion, and cell membrane permeability, thereby facilitating horizontal gene transfer (HGT) of ARGs. However, under stable operation at an OLR of 6.0 g·VS/L·d, there was a decrease in ARGs abundance but a notable increase in human pathogenic bacteria (HPB) and mobile genetic elements (MGEs). Subsequently, during reactor instability, the abundance of ARGs and HPB increased. Notably, during digestate recirculation at OLR levels of 6.0 and 7.0 g·VS/L·d, the process attenuated the risk of ARGs spread by reducing the diversity of ARGs hosts, minimizing interactions among ARGs hosts, ARGs, and MGEs, and weakening functional profiles associated with HGT of ARGs. Overall, digestate recirculation aids in reducing the abundance of antibiotics and ARGs under high OLR conditions. These findings provide advanced insights into how OLR and digestate recirculation affect the occurrence patterns of antibiotics and ARGs in dry AD.

Digestate recirculation is often considered an important way to improve system stability (system acidification, ammonia inhibition, hydrolysis limitations, etc.) and gas production performance. However, it is not clear how the promotion of biohythane production works in anaerobic co-digestion with digestate recirculation of rice straw (RS) and pig manure (PM). Two sets of laboratory-scale two-stage continuous stirred tank reactors were operated continuously for 95 d to investigate the performance of biohythane production in the first/second phase under mesophilic (M)/thermophilic (T) and digestate recirculation conditions. Firstly, biohythane was not produced by PM with RS under digestate recirculation. The main reasons were: 1) Digestive recirculation promoted the growth of hydrogenotrophic methanogenic bacteria; and 2) limitations in hydrolysis. Secondly, digestate recirculation has positive effects on the removal rates (removal rates of TS, VS, polysaccharide, protein and TCOD increased by 30.4%, 22.3%, 9.9%, 31.4%, and 11.9%, respectively) and energy yield (up to 68.7%). Finally, there was a higher abundance of hydrogen-producing bacteria (Fervidobacterium [44.9%] and Coprothermobacter [18.8%]) in T2, accounting for >80% of the total, and of which the huge hydrogen production potential cannot be ignored. The results provide new ideas for alleviating the energy crisis and developing green energy in the future.

The anaerobic co-digestion (coAD) of swine manure (SM) and rice straw (RS) is appealing for renewable energy recovery and waste treatment worldwidely. Improving its performance is very important for its application. In this study, long-term semi-continuous experiments were conducted to evaluate the improving effects of digestate recirculation on the performance, energy recovery, and microbial community of two-stage thermophilic-mesophilic coAD of swine manure (SM) and rice straw (RS). The experimental results indicated that the coAD systems of SM and RS (mixing ratio of 3:1) with or without digestate recirculation could not realize phase separation. The reactors of both coAD systems were characterized by pH values ranging from 7.74 to 7.85, methane production as 0.41 ± 0.02 and 0.44 ± 0.03 L/L/d, and stable operation. Notably, digestate recirculation increased total methane production, organic matter removal, and reaction rate of the coAD system by 9.92 ± 5.08, 5.22 ± 1.94, and 9.73-12.60%, respectively. Digestate recirculation improved the performance of the coAD by significantly increasing the abundance of Methanosarcina (from 4.1% to 7.5%-10.7% and 35.7%) and decreasing that of Methanothermobacter (from 94.2% to 87.3%-83.6% and 56.8%). Thus, the main methanogenesis pathway of the coAD system was changed by digestate recirculation and the methane production was effectively improved. Although the energy input of the coAD system increased by 30.26%, digestate recirculation improved the energy balance of the total system by 6.83%.

The effect of co-digestion of food waste (FW) and cow dung (CD) with different ratios, and digestate recirculation with different recirculation ratios (RR) on the substrate degradation and energy production in continuous two-stage anaerobic fermentation system was investigated. Results from experiments indicated that co-digestion and digestate recirculation could promote the hydrogen production rate (HPR) and the methane production rate (MPR). Maximum HPR and MPR of 3.3 and 3.1 L/L/d were achieved for two-stage fermentation with recirculation system at RR of 0.4. Meanwhile, both co-digestion and digestate recirculation technology could reduce the amount of alkali addition to maintain pH in the hydrogen-reactor. Compared to digestate recirculation, co-digestion of FW and CD promote much more energy production, 654.9 and 4854.8 kJ/kgVSr were obtained from the co-digestion of FW and CD with the ratio of 2:1 in the hydrogen reactor and the methane reactor.

Single-stage (S-N treatment) and two-stage anaerobic digestion with (T-R treatment) and without digestate recirculation (T-N treatment) for methane production using food waste (FW) were comparatively evaluated to examine the effects of digestate recirculation on anaerobic digestion (AD). Digestate recirculation positively affected the methane yield and organic loading rate (OLR). Metabolite correlation analysis revealed that a systematic hydrolysis degree of greater than 75% is crucial to achieve the complete recoverable yield of methane from FW. Digestate recirculation also markedly increased the system alkalinity, maintaining an optimum pH for methanogens. However, the ammonium accumulated by T-R treatment would destroy the metabolic balance between the hydrolytic bacteria and methanogens, especially at a critical OLR. Therefore, the appropriate control of two-stage AD systems with digestate recirculation is limited not only to OLR regulation but also to the prevention of ammonium accumulation.

Recirculation of digestate was investigated as a strategy to dilute the food waste before feeding to anaerobic digesters, and its effects on microbial community structure and performance were studied. Two anaerobic digesters with digestate recirculation were operated at 37 °C (MD + R) and 55 °C (TD + R) and compared to two additional digesters without digestate recirculation operated at the same temperatures (MD and TD). The MD + R digester demonstrated quite stable and similar performance to the MD digester in terms of the methane yield (around 480 mL CH4 per gVSadded). In both MD and MD + R Methanosaeta was the dominant archaea. However, the bacterial community structure was significantly different in the two digesters. Firmicutes dominated in the MD + R, while Chloroflexi was the dominant phylum in the MD. Regarding the thermophilic digesters, the TD + R showed the lowest methane yield (401 mL CH4 per gVSadded) and accumulation of VFAs. In contrast to the mesophilic digesters, the microbial communities in the thermophilic digesters were rather similar, consisting mainly of the phyla Firmicutes, Thermotoga, Synergistetes and the hydrogenotrophic methanogen Methanothermobacter. The impact of ammonia inhibition was different depending on the digesters configurations and operating temperatures.