Sequential batch thermophilic solid-state anaerobic digestion of lignocellulosic biomass via recirculating digestate as inoculum – Part II: Microbial diversity and succession
Introduction
Anaerobic digestion (AD) is a mature and common waste management technology that relies on microorganisms, both bacteria and archaea, to produce energy and reduce waste streams under oxygen-free conditions. Successful AD system performance primarily depends on the robustness of these microbes. Theoretically, the AD process involves four microbial metabolic steps: hydrolysis by hydrolytic bacteria that reduce substrate polymers to monomers; acidogenesis and acetogenesis by acid-forming bacteria that ferment monomers to volatile fatty acids (VFAs, primarily acetate), carbon dioxide (CO2), and hydrogen (H2) and other byproducts; and lastly, methanogenesis by methanogenic archaea that produce methane from the fermentation products, mainly acetate, CO2, and H2 (Li et al., 2011). Interactions among the diverse bacteria and archaea are complex and any imbalance may disturb or even cause the AD process to fail (Li et al., 2015).
Various AD systems and designs are available in terms of operating temperature, moisture, substrates, inoculation, and other options. Thermophilic AD (55 °C) has been commercialized widely in Europe for treatment of animal manure and the organic fraction of municipal solid waste due to its enhanced degradation of organic matter and destruction of pathogens (De Baere and Mattheeuws, 2012). Increases in the generation of solid wastes, including municipal and agricultural wastes, have fostered the development of solid-state AD (SS-AD) that operates at >15% total solids (TS) content (Li et al., 2011). High TS content allows a small reactor volume, low energy input for heating, and prevention of floating or stratification of solids. In recent years, thermophilic SS-AD has attracted much attention due to its potential to convert lignocellulosic biomass to energy more efficiently than mesophilic SS-AD (Shi et al., 2013). In a recent study, a sequential batch thermophilic SS-AD of yard trimmings was developed that exhibited long-term stability when digestate was recirculated as the inoculum (Lin and Li, 2017). That study was conducted for 4 runs, with each run lasting 30 days. Interestingly, the system was found to gradually reach steady state by the 3rd run with slightly higher methane yields (up to 11.5%) and cellulose degradation (up to 55%) than in runs 1–2. The results suggest that recirculation of the SS-AD digestate as the inoculum might have provided microbes that had been acclimated to the substrates and operating conditions. Characterization of the diversity and successions of the microbial communities of this SS-AD system will increase understanding of the sequential batch thermophilic SS-AD process.
Numerous studies have analyzed the microbial communities in AD systems. Although early studies used community fingerprinting methods, such as denaturing gradient gel electrophoresis (DGGE), fluorescent in-situ hybridization (FISH), and restriction fragment length polymorphism (RFLP) (Cabezas et al., 2015, Hori et al., 2006, Shi et al., 2013), next-generation sequencing (NGS) technologies have proved to be much more powerful in providing detailed characterization of communities of both bacteria and archaea in anaerobic digesters (Li et al., 2015). Recent studies using NGS revealed a vast diversity of both bacteria and archaea in AD digesters fed different feedstocks and operated under different conditions (Guo et al., 2014, Li et al., 2015, Yi et al., 2014). However, few studies have investigated how recirculation of solid digestate as inoculum affects the diversity and succession of bacterial and archaeal populations in thermophilic SS-AD systems treating lignocellulosic biomass.
The objectives of the present study were to: (1) determine the bacterial and archaeal diversity and succession in a sequential batch thermophilic SS-AD operation that recirculated digestate as the inoculum; and (2) study the correlation among the microbial community dynamics, environmental factors, and reactor performance. This study on microbial communities of SS-AD with sequential batch operation may also provide some insights for commercial scale systems. Besides, understanding the microbiology of SS-AD is not only critical for understanding the process itself, but also useful for improving and optimizing the process.
Section snippets
Sample information
The samples analyzed in the present study were collected in a previous study (Lin and Li, 2017). Briefly, a sequential batch thermophilic SS-AD system was developed by recirculating a portion of the digestate as the inoculum. Yard trimmings obtained from The Ohio State University’s Wooster campus (Wooster, OH, USA) were used as the substrates. The dewatered effluent (CTRL1) taken from a commercial mesophilic liquid anaerobic digester (L-AD, TS < 15%) was used as the initial inoculum to prepare
Summary of sequence data
In total, 1,195,520 quality-checked sequences were obtained for the 26 samples analyzed. The number of sequences for each sample ranged from 34,236 to 77,647 (45,981 on average) (Table 1). Additionally, 24,702 bacterial and 75 archaeal species-equivalent OTUs were found. Over 98.1% of all the sequences were assigned to the domain Bacteria, while about 1.4% of the sequences were assigned to Archaea, with the remaining unclassified. Nearly all of the bacterial sequences were classified to a
Conclusion
Extensive succession in bacterial and archaeal communities was observed during the sequential batch thermophilic SS-AD that recirculated digestate as the inoculum. The proportion of Firmicutes gradually increased, which might explain the improved cellulose degradation in runs 3–4. Dominant archaea shifted from Methanosarcina to Methanothermobacter during SS-AD, most likely due to VFA increases. This study showed that recirculation of digestate as the inoculum selectively enriched Firmicutes and
Acknowledgement
This project was funded by the USDA NIFA Biomass Research and Development Initiative Program (Award No. 2012-10008-20302) and USDA NIFA Hatch Program. The authors would like to thank Dr. Frederick C. Michel Jr. (Department of Food, Agricultural and Biological Engineering, OSU) for providing equipment for DNA extraction, Dr. Yueh-Fen Li (Department of Animal Science, OSU) for offering advice on sequence data analysis, and Dr. Harold M. Keener and Mrs. Mary Wicks (Department of Food, Agricultural
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