Analysis of energy recovery and microbial community in an amalgamated CSTR-UASBs reactor for a three-stage anaerobic fermentation process of cornstalks Open Access

In the last decades, with increasing energy demands and concerns about environmental impacts (Liu et al. 2022), the anaerobic fermentation process, which benefits from its efficient performance in waste and energy recovery (Wang et al. 2019a, 2019b), has been regarded as a viable way to produce bio-methane (around 50–70% of total gas) for replacing conventional energy sources (Cavinato et al. 2012; Baena-Morenol et al. 2019). Agricultural wastes, such as wheat and corn straws are abundant in carbohydrate contents, and are preferred renewable organic sources for the anaerobic digestion (Mahmoodi et al. 2018).

Two-stage anaerobic digestion (AD) process, which divides the digestion process into the hydrolysis and fermentation processes, has often been proposed as a feasible approach for producing bio-gas from various organic resources (Martinez-Perez et al. 2007). In a two-stage AD process, the degradation efficiency, energy recovery and stability of fermentation and methanogenesis phases were promoted (Luo et al. 2011). Pakarinen et al. (2009) obtained a higher hydrogen and methane production after NaOH pre-treatment step. Diamantis et al. (2014) also proved that two-stage CSTR-UASB system enhanced methane recovery as compared to the single-stage system. Co-digestion is another technology for improving the methane yield, which is also limited by the substrate supply (Wei 2016). Although two-stage AD could separate the whole digestion process into hydrolysis and gas production, the drawbacks of low energy recovery and low efficiency of chemical oxygen demand (COD) utilization still existed in recently studies (Wang et al. 2019a, 2019b). Therefore, further explorations are urgently needed to further advance the utilization and recovery radio of carbon in AD process.

The hydrolysis process of lignocellulose in cornstalks is considered as another rate-limiting step of the fermentation, which is one of the critical challenges of the anaerobic digestion of agricultural wastes (Cheng & Liu 2012). Although some methods, such as pretreatment of carbon resources (Wang et al. 2017), bioaugmentation (Zhang et al. 2015) and co-digestion (Almomani & Bhosale 2020), have been explored and applied to improve the fermentation process, shortcomings of the current technologies still need to be further studied. For instance, the traditional thermal and chemical pre-treatments need large amount of energy and chemical input, which also generated inhibitory, biologically non-degradable compounds and higher costs (Mulat et al. 2018). In addition, the chemicals residual would also produce inhibitory or toxic compounds (Fu et al. 2020). Therefore, developing simple and practical methods for pretreatment was significant.

In this study, a novel three-stage anaerobic fermentation reactor was explored for digestion of cornstalks. The innovation points of this study could be summarized as follows: (a) This reactor fully utilized the acidic environment in region A as a pre-treated process, replacing of the traditional steam-explosion or alkaline pretreatment (Mulat et al. 2018), for decomposing the lignocellulose of cornstalks. (b) There was no extra water supplied during experiments by intermittently suppling substrate without extra water added and recycling discharges for saving water resource and further reuse the residual carbon. (c) This study took advantage of CSTR for treating the high-density of solid cornstalks and UASB for transport liquid VFAs. As a result, the relative acid pH in region A was suitable for hydrolysis and VFAs produced and the relative alkaline pH was good for methanogenesis. The functional microbial communities and diversities in different districts were also discussed for high throughput sequencing for revealing the biological mechanism in this system.

The corn straw was harvested from Taigu farm, Shanxi Province, before being cut into 3–5 cm pieces after natural air-drying. The inoculated activated sludge was taken from the Zhengyang sewage treatment plant in Jinzhong City, Shanxi Province. The characteristics of the inoculum and substrate (tested in triplicate) are shown in Table 1.

Different functional (hydrolysis, acidification, and fermentation) sludge was enriched in the three regions to decrease the acidification effect on the methanogenesis process and the production of biogas residue/slurry and to increase the substrate utilization efficiency. The waste sludge was then discharged after the effluent re-entered the area through the backflow pipe for reaction until the high solids reactant reaction was complete. The sludge in regions B and C would be kept for the following high solid fermentation.

To incubate and start up the reactor, the number of corn cornstalks were periodically increased. The cornstalks were provided as 200 mg/L, 500 mg/L, and 750 mg/L at the operation time of 0–20 days, 21–40 days, and 41–130 days. The supplemented inorganic nutrients contained (mg/L): K₂HPO₄ (125), MgCl₂·6H₂O (15), FeSO₄·7H₂O (25), CuSO₄·5H₂O (5), CoCl₂·5H₂O (0.125), NH₄HCO₃ (5,240), and NaHCO₃ (6,720) (Endo et al. 1982). No extra water was added during the whole periods.

The concentrations of -N, -N, and -N were measured three times a day, following the standard methods (APHA 2005). Temperature and pH were measured by the temperature and pH meters (MT InPro6800, MT FE28, and MT Company). VFAs concentrations were determined using a gas chromatograph. TS and VS of the granular sludge were analyzed according to standard methods (APHA 2005). The concentrations of VFAs and methane were determined by Agilent 4004A gas chromatography.

DNA from the sample was sampled at the end of the whole experiment and extracted using the Bacteria DNA Isolation Kit Components (MOBIO), in accordance with the manufacturer's instructions. Polymerase chain reaction (PCR) amplification was performed as described in a previous study (Xie et al. 2020). The PCR amplification of the V4–V5 region of the 16S rRNA gene was conducted using bacterial universal primers 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) and 515F (5′-GTGCCAGCMGCCGCGG-3′). The compositions of the PCR products were identified by pyrosequencing on the Illumina Miseq sequencer platform (Shanghai Biozeron, China). SigmaPlot 14.0 was employ for raw sequences analysis.

The process of methane production by anaerobic fermentation is a complex process that requires the joint action of different microorganisms. Methanogens (Archaea) and acidogenic bacteria (bacteria) play a major role in the process of fermentation (Kong et al. 2018). Acidogenic bacteria provide methanogens with appropriate redox potential and fermentation substrate. Methanogens alleviate the inhibition feedback effect caused by early acid and hydrogen accumulation due to volatile acid depletion. Therefore, acidogenic bacteria and methanogens restrict and contact each other to complete anaerobic fermentation in this amalgamated CSTR-UASBs reactor. According to the Chao1 index analysis, The results indicated that, among the three zones of the reactor, zone A was the most active acid-producing zone and zone B was the most active methanogenic region. Therefore, during the stable operation of the reactor, the content of volatile acid in three chambers decreases in the order of A, B, and C.

At the genus level, as shown in Figure 6, Paludibacter (7.20%) was the dominant bacteria in region A, degrading cellulose and other macromolecules. Prevotella (15.31%) and Clostridium sensu stricto (10.23%) of bacteria were dominant bacteria in region B. Prevotella was closely associated with the production of volatile acids, degradation of lignin, cellulose, hemicellulose and other refractory macromolecular substances. Clostridium sensu stricto is a typical cellulose decomposing bacterium, that decomposes lignin, cellulose, and other volatile organic compounds after decomposition into monosaccharides (Lawson & Rainey 2015). The dominant bacterium in zone C was Caldisericum, accounting for 6.11% of the total bacteria. The main function of Caldisericum is to degrade sugars and produce volatile organic compounds.

The functions of bacteria in the three regions are roughly similar, but it can be seen that the sequence numbers of various bacterial communities vary considerably. The main function of area A is to degrade macromolecular substances such as cellulose and to produce volatile organic compounds by degrading sugars. Some of these substrates will be produced by hydrogen and carbon dioxide. However, this part of cellulose will not be completely degraded but will enter region B with the water flow. The main function of region B is to degrade cellulose and other substances. Cellulose degradation results in the production of volatile organic compounds, and a large number of cellulose degrading functional bacteria was detected in region B. Some sugars that have not yet produced volatile acid will enter area C, in the meantime. The microorganisms in region C will further decompose and produce acids. The generated volatile organic compounds will continue to circulate to zone A, where methanogenic reactions with water flow occur.

It can be seen from Table 3 that the dominant bacteria in region A are Methanobacterium (33.84%) and Methanthrix (10.66%). Methanobacterium is a genus of strictly anaerobic archaea and hydrogen trophic methanogenic bacteria, which mainly uses hydrogen and carbon dioxide as substrates for methanogenesis production (Zhu et al. 2017). Methanothrix is one of the most common methanogenic archaea with the methanogenic substrate is acetic acid, which cannot use hydrogen and carbon dioxide to produce methane. Methanobacterium and Methanthrix are also the dominant genera in region B. However, the content of Methanobacterium was 16.51%, which was lower than that in region A (33.84%). Methanothrix accounted for 27.28% of community genera, which was higher than that of region A (10.66%), because of the excess of acid in zone A, which led to the inhibition of acetic acid. Therefore, Methanobacterium, mainly utilizes hydrogen and carbon dioxide to produce methane and play a major role in methane production. After entering zone B, the concentration of volatile acid decreases, and methanthrix gradually replaces Methanobacterium as the dominant flora. Methanthrix remains the dominant species in region C, however the content of Methanolinea (18.1%) increased rapidly compared with region A and B. There are many metabolic pathways of Methanolinea, which can not only use hydrogen and carbon dioxide to generate methane, but also can use formic acid, acetic acid and other volatile substances to generate methane. This is due to the presence of hydrogen, carbon dioxide, acetic acid and other volatile acids in region C, the levels of which do not inhibit methanogens. Therefore, multiple methanogenic pathways can be carried out together, which contributes to the enrichment of Methanolinea.

Based on the microbial analysis, the main function of region A is the hydrolysis of straw cellulose and acid production of polysaccharides. The remaining cellulose flows into region B, where a large number of cellulolytic bacteria are enriched for cellulolysis. Some of the substrates produced after decomposition can be directly acidified, while other parts will be decomposed in in region C for acid production. Enrichment of hydrogen and carbon dioxide, as well as appropriate concentrations of acetic acid and other substances, diversify methane production.

In this study, a novel CSTR-UASBs hybrid reactor was successfully established for a three-stage anaerobic fermentation process of corn cornstalks. The results showed that the methane production rate could achieve 75.98% and methane produced from cornstalks was 520.07 mL/g. Paludibacter, Prevotella/Clostridium sensu stricto, and Caldisericum were detected the dominant bacteria of regions A, B, and C, responsible for the degradation of cellulose, lignin, cellulose, and other refractory macromolecular substance. Methanobacterium in regions A and B were mainly used for methanogenesis production, while Methanolinea can be used to produce methane gas from acetic acid and other organic substances.

This research was supported by the Ministry of Environmental Protection of the People's Republic of China (Major Science and Technology Program, No. 2019YFC0408601 and 2019YFC0408602), the National Natural Science Foundation of China (NSFC, No. 52070139; 52100155; 42177057), the Natural Science Foundation of Youth Fund of Shanxi Province (No. 202103021222009), and the Foundation Research Program of Shanxi Province, China (No. 202103021224099; 20210302124347).

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.