3.4.1. Performance of Bacterial Communities
There are 2331 bacterial OTUs classified into 24 phyla and 263 genera. Then, a large majority of them were classified into Firmicutes with about 89.2%. The average relative abundances of Firmicutes for T1, T2, T3, T4, and T5 reactors were 90.7%, 94.2%, 96.5%, 89.2%, and 92.5%, respectively (
Figure 4). This indicated that five reactors in this study may have achieved better digestion performance due to the predominance of Firmicutes and greater bacterial diversity [
26]. For the others, these were assigned to six phyla and others, which were Chloroflexi (2.6%), Proteobacteria (1.8%), Tenericutes (1.7%), Bacteroidetes (1.5%), Spirochaetes (1.5%), and Synergistetes (1.0%). Identical to previous studies, we found that Firmicutes and Chloroflexi were able to degrade a large number of organic compounds under a variety of conditions and had been found in a wide range of co-digestion [
26]. Illumina MiSeq results indicated that the uppermost bacteria were all affiliated to the phylum Firmicutes in the five reactors and in the control fed with different proportions of substrates. Members of Firmicutes have been reported as the dominant community in reactors [
26]. Moreover, most of the data in Firmicutes were attributed to the class Clostridia viz. 76.7%, 80.1%, 84.5%, 74.8%, and 77.0%. Only few of them were affiliated with the class Bacilli with 12.0%, 13.1%, 10.6%, 13.2%, and 14.1% in turn. Thus, the species of
Bacillus were significantly correlated with the concentration of protein in the reactor (
p < 0.01). The variation of ammonia nitrogen in different proportions during the anaerobic digestion process in
Figure 3b confirmed this relationship. T3 reactor with 60% protein and T5 reactor with 33.3% held a higher concentration of NH
4+-N than other reactors. The dramatic proliferation of Clostridia, known as hydrogen producer, prompted the generation of excessive hydrogen. Hydrogen partial pressure likewise affected fermentation performances and final product composition [
33]. It was reported that excessive hydrogen decreases hydrogenase enzyme activity and diverts metabolic pathways towards solvent production [
33]. T4 had the lowest relative abundance of
Clostridia with 74.8% and the highest BMP and GR, suggesting that more hydrogen was converted to methane. Moreover, Alibardi and Cossu [
33] reported that the carbohydrate content of substrates related to hydrogen yield little. However, the carbohydrate content in the reactor did not correlate to Clostridia abundance due to different proportions of carbohydrate, protein, and lipids in this study.
Microorganisms (>1.0%) mainly pertain to Chloroflexi, Proteobacteria, Bacteroidetes, and Spirochaetes, except for Tenericutes and Synergistetes that are those that had a trend decrease in relative abundance after the change of composition of substrate, as indicated by a similar report by another researcher [
34]. Chloroflexi found in T4 had the highest relative abundance (2.9%) in contrast to the other four reactors. Since Chloroflexi participates in carbohydrate degradation [
35], the higher the proportion of carbohydrate, the greater the abundance. However, the relative abundance of T2 with carbohydrates of 50% VS was 0.8%, less than four-fold compared to T4. It has been reported that some species belonging to Chloroflexi are associated with hydrogenotrophilic methanogens [
35]. However, Chloroflexi abundance was sensitive to pH fluctuation. The pH value of the T3 reactor fluctuated dramatically, resulting in a low relative abundance of Chloroflexi with 0.1%. On the other hand, the relative abundance of Tenericutes showed a significant relationship with the concentration of lipid (
p < 0.01). Furthermore, it was also correlated with TMP and DMP (
p < 0.05). With increasing lipid content, Tenericutes abundance kept growing, which was a failure indicator in AD with FW [
36]. Its abundance showed a correlation with C/N (
p < 0.05). Consequently, those changes inhibited the methanogens community and proceeded to the next step of methane product. The results indicate that there may be an interaction among substrates that could significantly affect the microorganism community.
Bacteria are responsible for the degradation for different composition proportions to intermediate metabolites. Those intermediate metabolites can be later utilized by methanogens.
3.4.2. Performance of Archaeal Communities
Both the bacterial and archaeal community structure was influenced by the co-digestion of different proportions of substrates [
37]. Furthermore, it was also reported that archaeal communities would be altered by bacterial populations [
26]. Thus, to help provide the required evidence, clarifying the relationship between archaeal community structure and composition proportions is necessary. The archaeal community at the end of fermentation were analyzed. The rarefaction curves of the six samples indicated that the sequencing depth for archaeal was sufficient to cover almost the whole diversity. A total of 6783 archaeal OTUs were classified into three phyla, belonging to Euryarchaeota, Crenarchaeota, Parvarchaeota, and others. For Euryarchaeota and Crenarchaeota, the relative abundances were 85.0%, 85.0%, 83.4%, 51.1%, and 85.1% and 14.9%, 14.8%, 16.3%, 48.6%, and 14.6%, for T1 to T5, respectively. A significant divergence was found among the six reactors where T4 was obviously different from other reactors, whose relative abundance was half of that of others for Euryarchaeota and three times greater for Crenarchaeota. For T4, the relative abundances of Euryarchaeota (51.1%) and Crenarchaeota (48.6%) was similar. This implied that the archaeal microorganisms had a balanced development, thus, achieving better coordination at the condition of T4 proportion substrates.
At the genus level, there were more than half of archaea reads, which matched unclassified microorganisms (62.5%). To facilitate the discussion, we classified genus except for unclassified on the basis of their relative abundance as: predominant (>5%), abundant (>1%), medium (>0.5%), rare (>0.01%), and very rare (≤0.01%) according to De Francisci et al. [
35]. Rare and very rare were not considered in this study. Relative abundances above 0.5% at genus level were used to build a histogram (
Figure 5). The predominant genera identified were
Methanosarcina,
Methanosaeta,
Methanoculleus, and
Methanobrevibacter originating from Euryarchaeota in the five reactors. Most of the CH
4 is produced by mainly two types, namely acetoclastic and hydrogenotrophic [
9]. According to Zabranska and Pokorna [
38], acetoclastic methanogens belong to
Methanosarcina and
Methanosaeta;
Methanobrevibacter and
Methanoculleus are the most commonly identified hydrogenotrophic methanogens. Taxonomic analysis showed that there were no notable changes of methanogens composition among different composition proportions but a distinct difference in the relative abundance of each genus.
Methanosarcina and
Methanosaeta affiliated with acetoclastic methanogens occupied 27.8%, 54.6%, 46.9%, 10.7%, and 30.2% and 3.3%, 2.8%, 6.5%, 3.0%, and 1.5% in the five reactors, respectively. This clearly indicated that methanogen abundance in T4 followed a tremendous difference compared to the others.
Methanosarcina in T4 had only 10.7%, which was below 2.6-fold in T1, 5.1-fold in T2, 4.1-fold in T3, and 2.8-fold in T5.
Methanosarcina, the main member in all the reactors, are mainly acetoclastic methanogens [
35] with the ability to sustain hydrogenotrophic and methylotrophic [
39] methanogenesis. Clearly, the substrate proportion affected the hydrolysis rate, which could result the relative abundance of
Methanosarcina. Especially,
Methanosarcina abundance was negatively correlated with BMP (
p < 0.05), which indicates that their lower abundance in the reactor would lead to more methane production in the T4 reactor.
Methanosaeta’s relative abundance was generally lower than that of
Methanosarcina in each reactor. Many studies demonstrated that
Methanosarcina and
Methanosaeta were competitive genera in AD [
26].
Methanosarcina is more tolerant to inhibitors than
Methanosaeta, e.g., it has a higher growth rate and tolerance to pH changes [
38]. Thus, the abundance of
Methanosaeta would change with varying pH during the digestion process. Its abundance was positively correlated with pH (
p < 0.05). Another reason is that
Methanosaeta can only use acetate for CH
4 production, but low concentrations of acetate favors its growth. Therefore, the result suggested that the T5 reactor had a higher acetate concentration and its two genera differed 20.1 times. Acetate supplied nutrition for
Methanosarcina metabolism and also significantly increased when
Methanosarcina increased [
26]. It is generally recognized that carbohydrates have the fastest hydrolysis. T2 had the maximum proportion of carbohydrate with 50% VS and the maximal
Methanosarcina relative abundance with 54.6%. However, its BMP and gasification rate were lower than those of T4. It has been reported that the AD rate is limited by methanogenesis rather than by hydrolysis due to the rapid acidification of substrates to VFAs, resulting in a rapid decrease in pH and process inhibition of methanogens activity [
38]. In fact, the pH of T2 was lower than the value during the corresponding period. Furthermore, this was mainly caused by the acid resistance of some
Firmicutes species, especially
Clostridium which can grow at low pH.
Universally, structure determines function. Archaeal community structure influenced methane yield via different relative abundance of acetoclastic methanogens and hydrogenotrophic methanogens (
Table 5). The relative abundance of the dominant population of two types of metabolic pathways is shown in
Table 5. Consequently, the two types of relative abundance of T4 reactor were 13.7% and 13.0%. Moreover, a negative correlation was found between actoclastic methanogens and BMP (
p < 0.05) in this study. T4 reactor composition proportion could buffer ammonia and VFAs to adjust the pH and reduce the inhibition of acidification to methanogens, while it could also balance the dominant population of the main two metabolic pathways. The similar relative abundance means that two types of methanogens could be supplementary and competitive to each other. These micro-environmental niches are in favor of methane yield and other products. Furthermore, the species richness, measured via OTUs’ rank abundance curve and chao1, suggests that the T4 reactor had the more abundant microorganisms and more diversity (
Table 6). The T4 reactor had an optimal internal micro-environment that coordinated the balanced development of various microorganisms. Due to the competitive, exploitative, and cooperative relationships, predominant microorganisms are enabled to balance the population in an attempt to take over the metabolic niche. This improved the diversity of the AD system, thus, ensuring the stability and increase of the methane yield.
Furthermore, this clearly indicates that composition proportions played essential roles in influencing the archaeal community structure. This affected the final methane yield and anaerobic products.