Biogas production performance
The AD performances of RS, cellulose, and hemicellulose are shown in Figure 1. The daily biogas production of RS and its main components contained two daily gas production peaks during the entire AD process (Figure 1A). The intensity of the first peak was lower than that of the second peak for RS digestion. In contrast, the first gas production peaks from cellulose and hemicellulose digestion were higher than the second. Among them, production peaks of RS digestion occurred on the first and sixth days, and biogas production on these days was 14.19 and 34.78 mL/g·VS-1, respectively. The two biogas production peaks of cellulose appeared on the third and twelfth days, and biogas production on these days was 85.36 and 35.64 mL/g·VS-1, respectively. The first biogas production peak of cellulose was 501.55% higher than that of RS. The second biogas production peak of cellulose was similar to the second gas production peak of RS. The two biogas production peaks of hemicellulose appeared on the fourth and fourteenth days, with production on these days of 45.00 and 30.00 mL/g·VS-1, respectively. The first biogas production peak was 217.12% higher than that of RS digestion, and the second biogas production peak was 15.71% lower. The peaks of biogas production of RS occurred significantly earlier than those of cellulose and hemicellulose. This may be because RS contains fructose and other substances, in addition to cellulose and hemicellulose, which are more anaerobically digestible[4], leading to the early start of the AD process.
For cumulative biogas production, cellulose had the highest at 620.64 mL/g·VS-1, followed by hemicellulose at 412.50 mL/g·VS-1, and RS at 283.75 mL/g·VS-1 (Figure 2B). The cumulative gas production of cellulose and hemicellulose was 118.72% and 45.37% higher than that of RS, respectively. These results are consistent with those of Li et al.[1, 16]. This is because the VS of RS is lower than that of cellulose and hemicellulose, which also contain lignin. Studies have shown that lignin is composed of three main monomers: coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol. These basic monomers constitute the basic structural unit of lignocellulose: syringyl phenylpropane (S), guaiacyl phenylpropane (G), and p-hydroxyphenylpropane (H)[20]. Lignin is challenging to biodegrade under anaerobic conditions[21, 22]. Indeed, in straw, cellulose, hemicellulose, and lignin form structures that are difficult to biodegrade [8, 9]. In addition, some cellulose and hemicellulose cannot be used by microorganisms in RS, so its cumulative gas production is significantly lower than that of cellulose and hemicellulose. Inhibition mechanisms may also contribute to the difficulty of biodegrading enzymes to adsorb to hydrophobic lignin[23].
For the methane content during AD, RS first showed a slow increase, reaching a maximum of 72.39% on the fifth day, and then gradually decreased to 61.44% on the eighth day, finally stabilizing at around 60% (Figure 1C). The trends for cellulose and hemicellulose were similar: both showed a slow increase in the early stage of DA and stabilized at about 70% after day 14. Before day 12, the methane content of RS was higher than that of cellulose and hemicellulose. After 12 days, the methane contents of cellulose and hemicellulose were higher than that of RS. After the methane content stabilized, the methane content of the biogas produced from cellulose and hemicellulose were approximately 10% higher than that produced from RS.
The pH level, ammonia nitrogen content, and total alkalinity are important indices that can be used to evaluate the stability of anaerobic digestive systems and microbial metabolism. The pH levels of the digestion sludge of RS, cellulose and hemicellulose were 7.33, 7.29, and 7.25, respectively, and the alkalinity levels were 4500, 4200, and 4100 mg/L, respectively. The values for each of these parameters were within the normal range: pH > 6.8 and alkalinity > 2000 mg/L[24]. In addition, the ammonia nitrogen contents of RS, cellulose, and hemicellulose were 710, 720, and 735 mg/L, respectively, which did not exceed the tolerance range of anaerobic microorganisms, which is 2 g/L. The methane production process was not suppressed[25], and the system reached a stable status after AD.
The modified Gompertz model was used to fit the dynamics of AD of RS, cellulose, and hemicellulose to the dynamics of biogas production. The correlation coefficient, R2, of the experimental value and the fitted value was above 0.98 (Figure 1D; Table 2). The maximum biogas production of the fitted value was very similar to the cumulative biogas production. The biogas production rates Rm of cellulose and hemicellulose were 266.79% and 144.09% higher than the Rm of RS, respectively, and hemicellulose showed the longest lag time.
Volatile fatty acids
VFAs are important intermediate metabolites in the process of AD and are one of the indicators that characterize the performance of AD[26]. Changes in VFAs during AD of RS, cellulose, and hemicellulose are shown in Figure 2. The accumulation of VFAs occurred in the early stages of AD of all three raw materials (Figure 2A). As AD proceeded, the accumulated VFAs were consumed. The accumulation stages of VFAs during AD of RS, cellulose, and hemicellulose were 4, 16, and 18 days, respectively. The concentration of VFAs from RS digestion was highest on day one (740.12 mg/L), while VFA concentration from cellulose reached its maximum on day seven at 2414.29 mg/L and was 3,26 times the highest concentration of RS. The maximum concentration of VFAs from hemicellulose was reached on day nine, which was 2234.58 mg/L and was 3.01 times the highest concentration of RS. The VFA concentration of RS stabilized below 50 mg/L from day five, and cellulose and hemicellulose stabilized below 50 mg/L from days 16 and 18, respectively. According to Lee et al.[27], VFA concentrations above 6000 mg/L suppress AD. As VFA concentrations in this study did not exceed 2500 mg/L, expectations were met in that no suppressive effects were seen. It has been reported that xylan in lignocellulose promotes the swelling of cellulose, thereby increasing its enzymatic hydrolysis[28], which may be the reason why RS enters a hydrolytic acidification condition earlier than cellulose and hemicellulose.
Studies have shown that acetic acid is the main carbon source for the AD of methane production, and other VFAs, including propionic acid, are also mostly converted into acetic acid to be inserted into methane metabolism by microorganisms[29]. The composition of VFAs is closely related to the performance of AD[30]. For the composition of VFAs produced during AD of the three raw materials (Figure 2B, C, D), the main component of the cumulative stage from day one to day four of RS was acetic acid (88.38%–91.88%). For cellulose, the acetic acid content was highest on day one (79.08%), and the main component for days 2–16 was propionic at 65.70%–98.42%. Similar results were observed for hemicellulose for days 1–18). Acetic acid (52.60%) dominated the first day, and propionic acid was the main component for days 2–18 at 81.57%–97.19%. Studies have shown that VFAs with even- and odd-numbered carbon are beneficial for the synthesis of 3-hydroxybutyrate and 3-hydroxyvalerate[31], respectively, while 3-hydroxybutyrate is more beneficial for the methanation process. The accumulation of substantial amounts of propionic acid could inhibit the AD process[31]. Studies by Pullammanappallil et al.[32] showed that the maximum accumulation of propionic acid was 2750 mg/L, which did not inhibit AD. In this study, the maximum cumulative concentrations of propionic acid of cellulose and hemicellulose were 2149.01 and 2148.60 mg/L, respectively, neither of which exceeded 2750 mg/L. Inhibition of digestion is thus not likely. When the AD progressed into the stable biogas production stage, and the methane content remained stable, the accumulated propionic acid was quickly consumed.
In this study, the concentration of VFA produced from the AD of RS reached a maximum value before cellulose and hemicellulose, but its maximum concentration was lower than that of the other two components. This finding is consistent with the law of biogas production that discussed before. Similar to VFAs, the biogas yield of RS was lower than that of cellulose and hemicellulose. The main component of VFAs that accumulated during the AD of RS was acetic acid, which was quickly consumed. The major component of VFAs that accumulated during the AD of cellulose and hemicellulose was propionic acid, which accumulated over an extended period. This finding is consistent with the law of methane content during the AD of these three materials, and it shows that the production of VFAs had a dual effect on the efficiency and biogas production performance of AD. On the one hand, VFAs are substrates for methanogenesis. Within a certain concentration range, the accumulation of VFAs is positively correlated with biogas production. On the other hand, the excessive accumulation of VFAs will also lead to the inhibition of AD.
Microbial community
After splicing all the sequences, quality control, and filtering out the low-quality sequences, a total of 175,423 bacterial sequences and 132,506 archaea sequences were obtained. The obtained bacterial and archaea sequences were clustered using the UPARSE algorithm, and a total of 534 bacterial OTUs and 21 archaea OTUs were obtained. These 34 bacterial OTUs belong to 31 different phyla, 60 different classes, 94 different orders, 142 different families, and 243 different genera. The 21 archaea OTUs obtained belonged to four different phyla, six different classes, seven different orders, 11 different families, and 15 different genera.
The composition of microbial communities at the genus level is shown in Figure 3. In the bacterial Venn diagram (Figure 3A), the data show that most of the bacteria in the three raw materials were the same, with 181 genera accounting for 74.49% of the total bacterial genera. The number of bacterial genera unique to the AD systems of RS, cellulose, and hemicellulose were 13, 9, 6, respectively. In the archaeal Venn diagram (Figure 3B), the data show that the archaeal genus in the AD systems of the three raw materials was the same.
As shown in the ternary phase diagram of the bacteria (Figure 3C), Clostridium is the dominant genus in the AD systems of RS, cellulose, and hemicellulose, with relative abundances of 38.44%, 43.14%, and 40.62%, respectively. The second most abundant genus was Terrisporobacter with relative abundances of 12.29%, 12.42%, and 11.65% for the AD systems of RS, cellulose, and hemicellulose, respectively. In addition, the abundances of Sedimentibacter and Romboutsia in the AD systems of the three raw materials are also very similar. Ruminofilibacter showed a higher abundance in RS AD and a lower abundance in cellulose and hemicellulose ADs. Bacteroidetes vadinHA17 and vadinBC27 wastewater sludge groups were present in the cellulose and hemicellulose AD systems in higher abundances than in the RS system. The abundance of Bacteroidetes vadinHA17 in the cellulose AD system was higher than in the hemicellulose AD system, and the abundance of the vadinBC27 wastewater sludge group in the hemicellulose AD system was significantly higher than in the cellulose AD system.
In the process of anaerobic acidification, carbohydrates are hydrolyzed into ethanol, lactic acid, acetic acid, and butyric acid by monosaccharide fermentation acid-producing bacteria, and lactic acid is further reduced to propionic acid. At the same time, VFAs are mutually converted by redox reactions under the action of different microorganisms. Clostridium plays an important role in degrading organic components and generating VFAs[33]. For example, Merlino et al.[34] found that Clostridium can degrade wood fibers and vegetarian raw materials. Terrisporobacter is a strictly anaerobic bacterium that can degrade carbohydrate raw materials into acetic acid[35]. In this study, the abundances of Clostridium and Terrisporobacter were relatively high, which provided a suitable environment for the smooth progress of the hydrolysis and acidification process in the AD system. It has been reported that the vadin BC27 wastewater sludge group produced small molecular fatty acids from fermentable amino acids, cysteine, leucine, methionine, serine, tryptophan, and valine[36], and was well adapted to ammonia nitrogen concentrations[37].
We found that the substrates available for methane production in our study were very limited. This is consistent with Methanosaeta, which can only use acetic acid as a substrate for AD [38] to produce CH4. Whereas, Methanobacterium produces methane through CO2 reduction with hydrogen[39], and Methanomassiliicoccus produces methane by reducing methanol[40]. From the ternary phase diagram of the archaea (Figure 3D), it is evident that Methanosaeta is the dominant bacterium in the AD systems of the three raw materials, with similar abundances of 81.66%, 79.24%, and 70.22% for the AD systems of RS, cellulose, and hemicellulose, respectively. Methanobacterium abundances were 5.39%, 4.23%, and 3.96%, and Methanomassiliicoccus abundances were 1.30%, 1.52%, and 1.74% for the AD systems of RS, cellulose, and hemicellulose, respectively, indicating that the AD of the three raw materials was based primarily on the reduction of acetic acid.
Functional Analysis
A total of 35.66 GB of data was obtained via high-throughput sequencing. Based on the original sequencing data, the low-quality and N-reads in the data were eliminated, and 331,859,324 high-quality sequences, required for subsequent analysis were obtained. 4,533,413 gene sequences were predicted using ORF prediction. When our results were compared with the KEGG database to obtain the functional annotations of each sample. The principles of the three raw materials at levels one, two, and three were found to be similar. At level one, metabolism was the most abundant pathway with relative abundances of 68.46%, 68.62%, and 68.56% for the AD systems of RS, cellulose, and hemicellulose, respectively. At level two, carbohydrate metabolism was the most abundant pathway, with relative abundances of 15.42%, 15.35%, and 15.29% for the AD systems of RS, cellulose, and hemicellulose, respectively. At level three, the most abundant metabolic pathway was biosynthesis of amino acids with relative abundances of 5.22%, 5.15%, and 5.15% for the AD systems of RS, cellulose, and hemicellulose, respectively, followed by carbon metabolism, with relative abundances of 5.04%, 5.11%, and 5.03 for the AD systems of RS, cellulose, and hemicellulose, respectively. The relative abundances of methane metabolism were 2.26%, 2.41%, and 2.29% for the AD systems of RS, cellulose, and hemicellulose, respectively. This is consistent with the findings of Li et al.[41] and the chemical composition of the raw materials.
At the module level, a total of four modules were related to methane production: M00357, M00567, M00356, and M00563. Of these, M00357 uses acetate as a substrate, M00567 uses H2-CO2, M00356 uses methanol, and M00563 is based on methylamine-dimethylamine-trimethylamine. The abundances of these four modules are shown in Table 3. M00357 accounted for the highest proportion of modules, with relative abundances of 47.10%, 47.62%, and 47.15% for the AD systems of RS, cellulose, and hemicellulose, respectively, followed by M00567 with relative abundances of 28.40%, 28.65%, and 28.98% for the AD systems of RS, cellulose, and hemicellulose, respectively (Table 3). The next was M00356, whose relative abundances were 23.07%, 22.51%, and 22.65% for the AD systems of RS, cellulose, and hemicellulose, respectively, and M00563 had the lowest relative abundances, which were 1.43%, 1.22%, and 1.21% for the AD systems of RS, cellulose, and hemicellulose, respectively. These results indicate that the methanogenic process in this study was aceticlastic methanogenesis, which is also consistent with the analysis results of microbial communities.
The functional composition at the enzyme level is shown in Figure 4A; it was compared with methane metabolism metabolic pathway map00680 to depict dominant pathways in this study (Figure 4B). The results in Figure 4 show that the most abundant pathway in this study is that acetic acid first produces acetyl-CoA, then undergoes a series of reactions to produce 5-methyl-5,6,7,8-tetrahydromethane, then biosynthesizes methyl-CoM, and finally produces methane.
In the methanogenesis pathway, the methyl coenzymes methyl-CoM and acetyl-CoA were important intermediate products. Five pathways can produce methyl-CoM. In this study, 5-Methyl-THM (S) PT produced the highest abundance of methyl-CoM through the action of enzyme EC 2.1.1.86, with relative abundances of 83.19%, 86.22%, and 86.14% for the AD systems of RS, cellulose, and hemicellulose, respectively.
Fisher’s exact test analysis in the STAMP difference analysis was used to compare the abundance of functions between two samples. Through this analysis, significant difference functions could be obtained. The gene set was constructed and annotated with methane metabolism and acetyl-CoA-related genes, and the abundance was calculated using the RPKM method. At the module level, the function of microorganisms in the AD systems of RS, cellulose, and hemicellulose were analyzed using STEMP. As shown in Figure 5, there is a significant difference between RS and cellulose in the acetyl-CoA related genes (Figure 5A). The results indicate that there are two deviations exceeding 1: the highest deviation is M00036, followed by M00088, with the deviations are +3.55 and -3.30, respectively. The difference between RS and hemicellulose in the acetyl-CoA-related genes (Figure 5B) was the same as that for cellulose. The two modules with the highest deviations were +3.33 and −2.76, respectively. The function of M00036 is to convert leucine to acetoacetate and acetyl-CoA, and the function of M00088 is to convert acetyl-CoA to acetoacetate, 3-hydroxybutyrate, or acetone. In the significant difference analysis of RS and cellulose in methane metabolism (Figure 5C), it was evident that the highest deviation was M00001, which is +0.50. Its function is to convert glucose to pyruvate from straw to hemicellulose. The significant difference analysis of RS and hemicellulose in methane metabolism (Figure 5D) showed that one of the absolute values of deviation was greater than 1 — M00001, the same as cellulose, with a deviation of +1.07. The results show that the AD process of RS, cellulose, and hemicellulose had little difference in the function of microorganisms during the methanogenesis stage, while the functions of microorganisms related to acetyl-CoA were quite different.