Cumulative biogas production of MAD increased steadily over time, which the daily biogas production remained at a relatively high level within 15 days (Fig. 1a), corresponding with increases up to 370.37ml/g VS and 313.04 mL/g VS of Lvzhou No. 1 and P. giganteum on the 30th days (Fig. 1b), respectively. Over the same period of 10 days, the biogas production (312.7 mL/g VS) of Lvzhou No. 1 in TAD reactor studied by Lei  was higher than that of our study (296.32 mL/g VS), but our biogas production was obviously higher than Lei's by the time of 30 days. What’s more, we also found that the biogas production fluctuated greatly in Lei's TAD process, so we guessed that its stability was not good enough. In the case of equal biogas production, our study of MAD has the advantages of good stability and low energy consumption. In terms of comprehensive economic efficiency, our MAD system was found to be feasible. What’s more, compared with previous studies on biogas production of MAD using traditional materials, the fermentation system of our study has higher biogas production [29–31]. This indicates that Lvzhou No. 1 and P. giganteum as promising energy grasses are more conducive to biogas production than traditional materials in MAD system.
MAD is actually a process in which organic materials were degraded by microorganisms to produce biogas. During the initial period of anaerobic digestion, macromolecular organic matter was consumed by active microbial communities; that is, complex organic matter such as carbohydrates, proteins and lipids were hydrolyzed into monosaccharides, amino acids and long-chain fatty acids, then these small molecules were acidified by fermentation bacteria into a mixture of VFA and other secondary products [32–33]. The VFA was relatively high on the first 10 days of fermentation, and the dynamic trend of pH value was inversely proportional to VFA. The VFA reached the peak on the second for Lvzhou No. 1 and third day for P. giganteum, respectively (Fig. 1e), the pH value reached its lowest value at this point (Fig. 1d). At the same time, the VFA was further converted by acetogenic bacteria into acetic acid, CO and H2, which provided direct substrates for the methanogens to produce CH4, H2S, CO2 and other mixed biogas . This was consistent with the result of 3.1, which confirmed that methanogens were relatively active in the first ten days of fermentation, and biogas production increased when VFA was decomposed by fermentation. The nutrients in the system were gradually consumed by bacteria during the anaerobic digestion process, resulting in a gradual decrease of VFA and a stable value of pH. In the absence of nutrients, methanogens were gradually inactivated to produce less biogas (Fig. 1a and Fig. 1b). Similar results were also reported by Huang, Ohemeng-Ntiamoah and Zhu [20, 35–36].
The bacterial groups at the phyla level for all samples of Lvzhou No. 1 and P. giganteum are referred to in Fig. 3. It was observed that Firmicutes and Bacteroidetes were predominant, followed by Proteobacteria, similar results were also previously reported [37–38]. Firmicutes and Bacteroidetes were found to be the main bacteria in anaerobic digestion [39–41]. The relative abundance of Firmicutes obviously increased from day 0 to day 1, this was caused by the increase in Clostridia-class species, such as Unidentified_Clostridiales and Romboutsia. Moreover, Clostridia-class species belong to Firmicutes phylum, and are responsible for the degradation of various substrates (sugars and proteins) as well as the production of VFAs . According to Spearman correlation, Unidentified_Clostridiales had a strong negative correlation with pH, which also indicated that Clostridium was an important bacterium in MAD.
As Bacteroidetes, the abundances of Lentimicrobium, Bacteroides and Roseimarinus were acetogenic bacteria that degrade large carbohydrate molecules to produce acid, which likely support the growth of methanogens [43–46]. Our result suggests that Lentimicrobium, Bacteroides and Roseimarinus had a higher abundance in the early and middle period of MAD. This indicated that a higher relative abundance of Bacteroidetes might result in a large number of organic acids to reduce pH and a high biogas production rate, which was in strong accordance with the results observed in this study (Fig. 5).
Proteobacteria have been reported to be the most dominant group in propionate-, butyrate-, and acetate-utilizing microbial communities in anaerobic digesters, and the high abundance of such group might lead to higher VFA consumption and biogas production . It was observed that the relative abundance of Proteobacteria was higher in the first five days, which led to the decrease of VFA and the increase of biogas production. In addition, this also well explains the decrease of VFA and biogas production in the later fermentation period.
From Spearman correlation, Desulfomicrobium was positively correlated with BC and MC (Fig. 5a). Desulfomicrobium, which uses sulfate or sulfonyl anion as terminal electron acceptor, sustains its own growth through the energy generated by anaerobic respiration and its product is H2S. Simple organic compounds (lactic acid, pyruvate, ethanol, formic acid, and hydrogen) act as electron donors for sulfate respiration. The sulfate which uses lactic acid as an electron donor does not fully breathe resulting in the formation of acetate and CO2. In this study, Desulfomicrobium was found to exist in the early and middle stages of biogas production (Fig. 3b), this was the main cause of H2S and CO2 in biogas .
The Euryarchaeota was the most dominant archaea phyla in MAD for all samples of Lvzhou No. 1 and P. giganteum (Fig. 4a). Euryarchaeota mainly includes several bacterial genera (Methanogens, Halobacterium sp., Hyperthermophiles) in ecological niches, while methanogens play an important role as the dominant archaea in the biogas fermentation system . During the methanogenesis process, methanogens can use acetic acid (such as acetic acid, formic acid, H2/CO2, methanol) produced from the hydrogen phase to produce CH4 [50–51]. Methanosaeta was predominant with a higher relative abundance in the genus level (Fig. 4b). Methanosaeta may be the most important methanogenic microorganism in nature, and they live in waste water, swamps and wetlands . Our result was consistent with the study about methanogenic archaea conducted by Gaspari M . In conclusion, methanogenic archaea played a very important role in MAD reactors.