B. papyrifera was ensiled with Lactobacillus plantarum (10⁶ cfu/g of fresh weight) and packed into vacuum-sealed plastic bags (dimensions 225 mm×350 mm). A total of 10 bags were made and stored at ambient temperature for 45 days. Following ensiling, 4 bags were randomly opened and completely mixed for compositional analysis and anaerobic fermentation experiment. Lactobacillus plantarum used in the present study was purchased from Lallemand Inc. (Quebec, Canada), which was previously used in an earlier report of our group [13].

The SE pretreatment was performed using laboratory scale equipment (QB-200, Gentle bioenergy Co., Ltd., Henan, China) with a capacity of 2,500 g of samples per batch as previously described by Chang et al [14]. In order to obtain optimal SE conditions, a serial of preliminary experiments with different reaction pressures (1.2 and 1.4 Mpa), cooking times (60, 80, 100, 120, 140, 160 s), and temperatures (150°C and 194°C) were conducted. According to the recommendations of other researchers [7, 14], the optimal SE parameter of B. papyrifera was selected to 100 s under 1.2 Mpa with a temperature of 194°C. The steam-exploded B. papyrifera samples were stored in sealed plastic bags for the anaerobic fermentation experiment. In addition, a portion of B. papyrifera without any physical or chemical pretreatments was used as control treatment.

Gas production was recorded before incubation (0 h) and after 1, 3, 6, 9, 12, 24, 36, 48, 60, and 72 h of fermentation. Four bottles were randomly selected from each treatment for determination of methane production at 12, 24, and 72 h of incubation using a gas chromatograph (GC-8A, Shimadzu Corp., Kyoto, Japan) with a flame-ionization detector. The temperature of steel capillary column (Porapack Q, 80/100 mesh, Waters Associates Inc., Milford, MA, USA) was maintained at 65°C and ultra-high purity nitrogen (99.999%) was used as the carrier gas at 40 mL/min flow.

The incubation fluid from each treatment was used for measurement of fermentation parameters. The pH of the incubation contents was determined using a digital pH meter (Hanna, Hi 8520, Ronchi di Villafranca, Italy). The VFA concentration and the molar VFA profile including acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate was measured using GC (Agilent Technologies, Palo Alto, CA, USA) with a capillary column (BP21, 30 m×0.25 mm ID×0.25 μm; Kinesis Inc., Vernon Hills, IL, USA). Lignocellulosic degradation of B. papyrifera samples were determined using the Daisy II incubator system for 72 h of incubation (ANKOM Tech., Fairport, NY, USA). This reaction unit consisted of four incubation jars containing 1.6 L of buffer solution and 0.4 L of rumen liquor with 25 nylon bags inside. Buffer formulation was based on Maggiolino et al [17] study and included 1.33 L buffer A and 266 mL of buffer B. Before the commencement of the incubation, nylon F57 filter bags (ANKOM Tech., USA) were rinsed in acetone and dried at 100°C for 24 h. Reprehensive samples from three pretreatments were dried in an air-force oven at 65°C for 48 h and ground through a 1-mm screen (Wiley mill, A. H. Thomas, Philadelphia, PA, USA) for incubation. The percentage loss in weight was measured as in vitro degradability at 24 and 72 h.

Methanogenic community composition in the incubation fluids (at 6, 12, 24, and 72 h of incubation) was examined by using high-throughput sequencing technique targeting the functional gene of methyl-coenzyme M reductase A (mcrA). The total genomic deoxyribonucleic acid was extracted with a QIAamp Fast DNA Stool Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. The real-time polymerase chain reaction (PCR) primers used to amplify the mcrA fragments was designed according to the method of Luton et al [18]: 5′-GGTGGTGTMGGATTCACACAR TAYGCWACAGC-3′ and 5′-TTCATTGCRTAGTTWG GRTAGTT-3′. The whole reaction was performed by using the TaKaRa rTaq DNA Polymerase system, which included a 2 μL of 10×buffer, 2 μL of deoxynucleotide triphosphate (dNTPs) mixture (2.5 mmol/L), 0.2 μL of rTaq polymerase, and 0.8 μL of each primer (forward and reverse). The PCR amplification was initiated at 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 45 s, and finished with a final extension of 72°C for 10 mins by using a 2700 GeneAmp PCR system (Applied Biosystems, Foster, CA, USA). Amplicons were then extracted from 1% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s instructions and were quantified using QuantiFluor-ST (Promega, Madison, WI, USA). Library was quantified and analyzed using the Agilent 2100 Bioanalyzer and the ABI StepOnePlus Real-Time PCR system. The qualified libraries were then sequenced on the Illumina HiSeq platform according to standard protocols. The raw sequence data were processed, and the quality filtered sequence were clustered into operational taxonomic units (OTUs) according to the 97% sequence similarity using QIIME.

After sequencing analysis and data processing, the average number of valid sequences was 18,755±3,178.5, 20,220±4,346.8, 17,298±4,102.6, and 18,991±3,657.5 for each incubation time (6, 12, 24, and 72 h), respectively. The Good’s coverage over 0.999 of all samples indicated that most common OTUs were identified and included into the analysis. Richness and diversity indices of methanogenic communities are shown in Figure 4. The Shannon indices did not change significantly among the three pretreatments throughout the incubation, whereas ensiled or steam-exploded pretreatment showed higher Simpson values than untreated material when incubated for 12, 24, or 72 h (p<0.05). In addition, steam-exploded pretreatment significantly enhanced the richness of the methanogenic community as Ace and Chao indices were highest among the three treatments at 24 and 72 h (p<0.05). These results demonstrated that ensiled or steam-exploded pretreatment enhanced diversity and richness of the methanogenic community during the incubation. Li et al [11] suggested that the improved biodiversity may be ascribed to the positive effects of methanogenesis and more accessible substrates for the growth of anaerobic microbes and methanogens. This conclusion was in line with our findings that increased VFA production was observed for ensiled or steam-exploded of B. papyrifera, which was beneficial to the substance degradation and high incubation performance.

Taxonomy analysis of methanogenic communities at the genus level is shown in Figure 5. Three major genera of Methanobrevibacter, Methanosarcina, and Methanosphaera were identified within the whole archaeal sequences in all samples. Methanobrevibacter was the most dominant genera with a relative abundance of 68.9%, 63.9%, 67.1%, and 59.4% when incubated for 6, 12, 24, and 72 h, respectively. This result was similar to the findings from Dong et al [26] who reported Methanobrevibacter accounted for 48.4% to 56.4% among methanogenic genera in ruminal samples. It was reported that sequences belonging to Methanobrevibacter accounted for approximately 62% of rumen archaea, with most of sequences relating to Methanobrevibacter gottschalikii (33.6%) and Methanobrevibacter ruminantium (M. ruminantium; 27.3%). In conformance with previous results, Methanobrevibacter sp. SM9 and M. ruminantium species were identified in the present study using the functional gene of mcrA sequencing technique, which accounted for 38.6% and 17.8%, respectively. Methanosarcina population was the second most abundant methanogen at each incubation period and was reported to be a multifunctional methanogen that could utilize either hydrogen/carbon dioxide or acetate for methane production [8,12]. Theuretzbacher et al [9] reported that increased abundance of hydrogen-utilizing methanogens during fermentation was associated with high levels of dissolved organic matter. It was also reported that increased abundance of Methanosphaera represented enhanced consumption of hydrogen, which would consequently reduce the H₂ accumulation and contribute to conversion of lignocellulose biomass to VFA [12].

The methanogen community structure was dynamic among the three treatments (Figure 5). Steam-exploded pretreatment consistently showed a higher population of Methanobrevibacter during the incubation but relatively low abundance of Methanosarcina when compared with ensiled pretreatment. The order of Methanosarcinales (e.g., Methanosarcina barkeri and Methanosarcina mazei) can use methanol and methylamines as substrates, but also metabolize acetate for methanogenesis. However, although both genera contributed to the methane generation, the metabolism activity and substrate utilization and conversion is still unclear. In addition, a small proportion of Candidatus Methanomethylophilus was observed among the three pretreatments when incubated for 24 and 72 h. Dong et al [26] reported a low abundance of Candidatus Methanomethylophilus (5.8%) in ruminal fluid and found that it was correlated with propionate concentration.

Methane formation is the final product of methanogenic archaea that use hydrogen or other substrates during the anaerobic fermentation of lignocellulosic biomass. Meanwhile, production and accumulation of VFA during incubation can reflect microbial activities and methanogenesis function. In the present study, the correlation matrix between the relative abundance of methanogen and fermentation parameters and methane production were examined by Spearman’s correlation heatmap at the species level (Figure 6). The correlation analysis showed that M. ruminantium was positively correlated with acetate, butyrate, total VFA concentration, and methane production when incubated at 6 or 24 h. Previous studies showed that Methanobrevibacter is the most abundant rumen archaea, with sequences mainly being associated with M. ruminantium (28%) and M. gottschalkii (33.6%) [27]. As the main hydrogen-utilizing methanogen, the abundance and activity of M. ruminantium is significantly affected by the production of acetate and butyrate, which is accompanied by the hydrogen formation during incubation. In addition, based on the complete genomic sequence analysis of M. ruminantium conducted by Leahy et al [27] it was suggested that M. ruminantium may be one of the protozoa-associated methanogens, and it can effectively obtain hydrogen for methanogenesis through hydrogen transfer from protozoa [26]. Meanwhile, positive correlation was also observed between butyrate concentration and Methanobrevibacter sp. D5 and Methanobrevibacter sp. SM9, which may be explained by the similar methanogenesis pathway and central metabolism to M. ruminantium. Methanosphaera sp. ISO3-F5 was reported at a low relative abundance accounting for an average abundance of 8.2%±6.7% in the rumen of cattle and sheep [26]. As one of the main methylotrophic methanogens, Methanosphaera sp. ISO3-F5 can utilize hydrogen to convert methanol into methane instead of reducing carbon dioxide to methane. In the present study, acetate was positively correlated with Methanosphaera sp. ISO3-F5, which would result from the increased availability of hydrogen when acetate formed during lignocellulosic degradation. Moreover, acetate concentration was positively related to the abundance of Methanosarcina barkeri and Methanosarcina Mazeiin in the present study, which was in agreement with the previous studies that acetate and other fermentation products (e.g., methanol and methylamines) were main substrates for the methane production by these two methanogen strains [24]. In conclusion, this study demonstrated that ensiling and SE pretreatments have a great potential for effective degradation of B. papyrifera. Compared with ensiling pretreatment, SE pretreatment decreased the ADF and NDF contents by 39.4% and 10.6%, respectively, after 72 h incubation. These results were closely related to the dynamic variations of methanogenic diversity and relative abundance. It is concluded that SE pretreatment may be a promising technique for the efficient utilization of B. papyrifera, contributing to sustainable livestock production systems