Fate of antibiotic resistance genes during high-solid anaerobic co-digestion of pig manure with lignite
Introduction
Antibiotics have been widely used for livestock industry, resulting in the high levels of antibiotic residues in animal manures (Burch et al., 2017). The antibiotic residues have created an environment of high selective pressure for antibiotic resistant organisms, which results in massive amounts of antibiotic resistance genes (ARGs) (Wichmann et al., 2014). Therefore, animal manure has become a typical reservoir of ARGs and mobile genetic elements (MGEs) in the environment (Hu et al., 2016, Wallace et al., 2018). Land application of animal manure as organic fertilizers can release a substantial amount of ARGs into the soil ecosystems, which is in risk of migration into the human food chain (Zhang et al., 2019d). The increasing dissemination of ARGs to the environment has become a major public concern, because resistance to antibiotics seriously reduces the effectiveness of antibiotic-based therapies and raises substantial risks to human health (Larranaga et al., 2018).
Previous studies found that there were high abundance and diversity of ARGs in pig manure (PM) from farms (Lu et al., 2017). Anaerobic digestion is a widely used method to produce fertilizer and biogas from livestock wastes (Zhang et al., 2016), and it is assumed that anaerobic digestion could contribute to the removal of ARGs (Song et al., 2017). The absolute abundance of ARGs was significantly decreased during anaerobic digestion of PM with wheat straw (Song et al., 2017), while some other studies reported an increase of some ARGs after anaerobic digestion (Ma et al., 2011, Zhang et al., 2016). In order to improve the efficiency of ARGs removal, additives can be added into the substrates of anaerobic digestion. For instance, addition of zero valent iron enhanced the potential of ARGs removal during anaerobic co-digestion of waste sludge and kitchen waste (Gao et al., 2017); but higher dosages of arsanilic acid and CuO nano-particles contributed to the propagation of ARGs during anaerobic digestion (Sun et al., 2017, Huang et al., 2019). Activated carbon and biochar are amorphous carbonaceous materials with high porosity and large surface area, and they play an important role in ARGs removal, and mitigation of ARGs spread (Zhang et al., 2018, Sun et al., 2018). The absolute abundance of some ARGs such as tetA and tetX decreased during anaerobic digestion of solid organic wastes with activated carbon (Zhang et al., 2018). The addition of 20 g·L-1 biochar significantly decreased the relative abundance of ARGs during anaerobic digestion of cattle farm wastewater, possibly through impacting the relative abundance of Firmicutes and Proteobacteria (Sun et al, 2018). However, the addition of activated carbon increased the relative abundance of tetM, tetW and tetO in digestion products of food waste with chicken manure (Zhang et al., 2018).
Lignite is a natural adsorption material (Li et al, 2015), and has rich pore structure and a high content of surface functional groups. Cellulose, lignin, humins or humic acids are commonly present in lignite, which could be degraded into methane by microbial activity under anaerobic conditions (Li et al., 2015, Detman et al., 2018). The C/N ratio of lignite is approximately 50–80 (Li et al, 2015), and therefore addition of lignite in PMs could enhance C/N ratio of the substrates and benefit the process of methane production. Lignite could provide sufficient habitats and nutrients for the growth of microorganisms which play an important role in enhancing the performance of anaerobic digestion (Li et al., 2015, Detman et al., 2018). The ARGs removal during anaerobic digestion is associated with the ARGs-carrying microorganisms, and therefore the changes of microbial community structure influenced the fate of ARGs (Zhang et al., 2018, Sun et al., 2018).
High-solid anaerobic digestion has high total solid (TS) (≥15%), which has many advantages such as smaller reactor volume, less energy input for heating, and minimal material handling (Zhang et al., 2015) compared with low-solid anaerobic digestion (TS < 15%). Moreover, high-solid anaerobic digestion of cattle manure significantly reduced ARGs and MGEs, and the total abundance of ARGs was lower in the mesophilic high-solid anaerobic digestion product than that in the thermophilic high-solid anaerobic digestion product (Sun et al., 2019). Therefore, high-solid anaerobic digestion (HS-AcoD) of PM with lignite is probably a promising approach to enhance the performance of anaerobic digestion on methane production and ARGs removal. However, to the best of our knowledge, no studies have investigated ARGs removal during HS-AcoD of PM with lignite.
In this study, by using high-throughput qPCR (HT-qPCR) with 296 validated primer sets, and high-throughput Illumina sequencing, we characterized the profiles of ARGs, MGEs and microbial communities during HS-AcoD of PM with lignite. The main objectives were to (1) investigate the effects of HS-AcoD of PM with lignite on removal of ARGs and MGEs, and to (2) explore the underlying mechanisms of removal of ARGs during HS-AcoD. We hypothesized that lignite may impact the fate of ARGs during anaerobic digestion through two possible mechanisms: (i) lignite may adsorb antibiotic residues in PMs and thereby reduce the selective pressure for microbial communities; and (ii) lignite supplies nutrients for the growth of microorganisms in anaerobic digestion systems, and indirectly influences the ARGs profiles through changing the microbial community structure.
Section snippets
Inocula and substrates
The inocula were collected from a large scale anaerobic digester and PM was obtained from piggery at Berry Bank farm (located at Windermere, Victoria, Australia), which had a standing swine population of 20,000 pigs. Lignite was collected from Bacchus Marsh coal mine in Australia. The inocula were centrifuged to enhance the content of microorganisms per unit volume before use, and the TS and volatile solid (VS) of the centrifuged inocula were 15.27% and 73.40%, respectively. The TS and VS of PM
Changes in cumulative methane yield
The inhibition of cumulative methane yield was observed at the initial stage of HS-AcoD, but at the medium stage of day 12, the cumulative methane yield of T1 and T2 increased by 36.52% and 18.44% compared to CK, respectively. At day 30, the cumulative methane yield for T1 increased by only 3.98%, while the T2 and T3 decreased by 7.71% and 28.71% compared to CK, respectively. During the entire HS-AcoD process, there was consistent inhibition of the cumulative methane yield for T4, possibly due
Conclusions
This study demonstrated that the addition of lignite can reduce the absolute abundance of ARGs and MGEs during HS-AcoD. The tnpA-03 gene might play an important role in the fate of ARGs during HS-AcoD of PM with lignite, followed by the IS613 gene. Lignite reduced the abundance of ARGs by reducing the microbial abundance and MGEs abundance. The 16% lignite addition could significantly decrease the abundance of ARGs and meanwhile maintain the HS-AcoD process. Thus, the addition of 16% lignite is
CRediT authorship contribution statement
Hai-Gang Guo: Conceptualization, Methodology, Data curation, Formal analysis, Visualization, Investigation, Writing - original draft, Writing - review & editing. Qin-Lin Chen: Conceptualization, Investigation, Writing - review & editing. Hang-Wei Hu: Conceptualization, Investigation, Writing - review & editing. Ji-Zheng He: Supervision, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by National Natural Science Foundation of China (41907206), Australian Government Cooperative Research Centres Projects (CRCPSEVEN000149), Australia-China Joint Research Centre of Healthy Soils for Sustainable Food Production and Environmental Quality (ACSRF48165), and Key Research and Program of Hebei (18237301D). We acknowledge the Melbourne Trace Analysis for Chemical, Earth and Environmental Sciences (TrACEES), The University of Melbourne for analytical support.
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