Acetate production
Acetate production performance of the MES reactors are shown in Fig. 1a. The cumulative acetate concentrations of the control in three batches were 88.2 mg/L, 92.4 mg/L, and 119.3 mg/L, respectively. The gradually increasing acetate production indicated the developing performance of the reactors. By supplement of ferrous and nickel ions, acetate production was significantly improved. The cumulative acetate concentrations of Fe group were 121.11 mg/L, 145.02 mg/L, and 186.51 mg/L, respectively in the three cycles, which were 37%, 57%, and 56%, higher than that in the control. The accumulated acetate concentration in cycle 3 of the Fe group was 79% higher than that before Fe2+ addition (Figure S1). The promotion of nickel ion addition was even better than ferrous ion addition. The cumulative acetate concentration of Ni group were 187.69 mg/L, 222.28 mg/L, and 264.13 mg/L, respectively, in the three cycles, and 113%, 140%, and 121% higher than that of control. The accumulated acetate concentration in cycle 3 of the Ni group was 118% higher than before Ni2+ addition, much higher than that in the control (49%). The significant improvement of acetate synthesis of the MES reactors by Fe2+ and Ni2+ addition was observed.
With the improved acetate synthesis, the electron recovery efficiency was also significantly increased with Fe2+ and Ni2+ addition (Fig. 1b). The electron recovery efficiency for the acetate production of the Fe group were 47.47%, 47.72% and 68.3%, respectively, in the three cycles, substantially higher than that of control (28.9%, 32.6% and 45.9%). With nickel ions addition, the electron recovery efficiency for the acetate production was enhanced to 63.44%, 75.40%, and 86.49%, respectively. Acetate synthesis was aided by the addition of Fe2+ and Ni2+, which also resulted in an increase in electron recovery efficiency, showing that the addition of Fe2+ and Ni2+ were favorable to the flow of electrons to WL pathway and acetate synthesis.
Microbial Community Of The Biocathode
The concentration of extracted total DNA and the microbial sequencing diversity of biofilms of the biocathodes in different groups are shown in Table 1. The concentration of total DNA extracted from the biocathodes of control, Fe and Ni groups were 531.3 ng/µL, 587.5 ng/µL, and 397.7 ng/µL, respectively, indicating the biomass on the cathodes of the Fe group was the highest, while that in the Ni group was relatively lower than the control. SEM also showed the relatively abundance biomass on the cathode of Fe group, along with many precipitates on the cathode surface (Figure S2). On the surface of the cathode from Ni group, biomass along with some precipitates were also observed. However, no precipitate was observed on the cathode of the control group. The abundant precipitates on the biocathode of Fe and Ni group were probably caused by deposition of added ferrous and nickel ions during operation. The ferrous ion and nickel ions concentration did drop gradually during the batch operation, and the precipitates were identified as the crystals of iron and nickel contained compounds (Figure S2).
Table 1
The concentration of total DNA and microbial sequencing diversity of different startup strategies among several indices.
|
concentration of extracted total DNA(ng/µL)
|
Shannon
|
Simpson
|
Ace
|
Chao 1
|
Coverage
|
Control
|
531.30 ± 2.50
|
2.77
|
0.10
|
127.61
|
129.33
|
0.999
|
Fe
|
587.50 ± 4.10
|
2.43
|
0.18
|
140.74
|
138.88
|
0.999
|
Ni
|
397.70 ± 3.40
|
2.99
|
0.08
|
148.10
|
155.11
|
0.999
|
The sequencing coverage of each sample reached 99.9%, indicated that the sequencing results were representative (Table 1). The Ace index and Chao 1 index were the highest in Ni group, and the lowest in the Control, which indicated that the richness of microbial community increased with the addition of Fe2+ and Ni2+. However, the Shannon index and the Simpson index showed that the microbial diversity of Ni group was the highest and the Fe group was the lowest, a relatively higher and lower microbial diversity in Ni group and Fe group than that in the control group were revealed.
Venn map (Fig. 2a) showed that the three groups had 85 shared OTUs, which accounted 89.4% − 98.9% of the total sequences in each library. The high proportion of shared OTUs and sequences indicated the similar microbial communities in the three group reactors. The main phyla in the three group reactors were Synergistetes, Proteobacteria, Firmicutes, Desulfobacterota, Bacteroidetes and Thermotogae, among which the total proportion of Synergistetes, Proteobacteria, Firmicutes and Desulfobacterota accounted for more than 80% (Fig. 2b). Fe group showed the increased relative abundance of Synergistetes, Proteobacteria and Firmicutes, and decreased the proportion of Desulfobacterota. Meanwhile, Synergistetes was increased by Ni2+ addition.
The main genera in the three group reactors were Desulfovibrio, Rhodocyclaceae, Aminobacterium, Blvii28_wastewater-sludge_group, and Clostridium_sensu_stricto_1, with total proportion of more than 60% (Fig. 2c). Relative abundance of Desulfovibrio and Rhodocyclaceae in control were 27.8% and 18.6%, respectively. In the Fe group, the relative abundance of Desulfovibrio decreased to 7.4%, while the relative abundance of Rhodocyclaceae increased to 34.7%. A large part of Desulfovibrio and Rhodocyclaceae are H2 producers (Perona-Vico et al. 2020; Xiang et al. 2017). The low potential of the cathode is beneficial to the survival of H2 producers, and resulted in the relatively high abundance of Desulfovibrio and Rhodocyclaceae. The relatively high abundance of Desulfovibrio and Rhodocyclaceae might affect the H2 production capacity of biochatode, and therefore promote the acetate synthesis with H2 mediated (indirect electron transfer). Overall, Fe2+ and Ni2+ addition had little effect on phylum level, and caused small changes in genus-level composition.
Metatranscriptomic Analysis
Basic information of metatranscriptomic libraries
Total 152 million clean reads (approximate 20 Gb) were produced from the three group MES reactors based on metatranscriptomic analysis (Table S1). The percent of non-r RNA in raw reads were 92.83% (control), 92.52% (Fe group) and 80.57% (Ni group), respectively. The number of transcripts obtained after splicing the reads were 62315 (control), 39450 (Fe group) and 59291 (Ni group), respectively. The obtained open reading frames (ORFs) were 46280, 28353 and 44429, from the control, Fe group, and Ni group, respectively.
The top 20 expressed genera indicated by metatranscriptomic data are shown in Fig. 3. Azonexus, Methanobacterium, and Desulfovibrio were the three genera with the highest transcriptomic expression. Acetogen genera, such as Clostridium and Acetoanaerobium, located in the top 20 expressed genera, though they were not the highest. Other high expressed genera included Mesotoga, Geobacter, Williamwhitmanis, Thauera, Aminobcaterium, Dechloromonas, Pseudomonas, and so on. The expression of Clostridium, Desulfovibrio, Williamwhitmania, and Aminobacterium were relatively high in Ni group, while the expression of Azonexus, Thauera, Dechloromonas, Pseudomonas, Acetoanaerobium, Malikia, Burkholderia, and Perimonas was the highest in the Fe group. Significant inhibition of expression of Methanobacterium, as well as Methanobrevibacter, by Fe2+ and Ni2+ addition was observed. The expression inhibition of Methanobacterium and Methanobrevibacter by Fe2+ addition was higher than that by Ni2+ addition. Fe2+ addition and Ni2+ addition showed different effect on the metatranscriptomics of the microbial community in the MES. The generation of methane was inhibited by the BES, however, the large amounts of the methanobacteria were found in the cultures. The BES is a structural analog of coenzyme M that involves in the final step of methane biosynthesis. The transcriptional expression of upstream genes may still occurred, and the growth of methanobacteria may not inhibited by BES. Therefore, the methanogens still existed in the reactors with methanogensis inhibited by BES.
Gene Annotation
The distribution of transcripts to general functional categories was assessed based on the BLAST against KEGG database (Fig. 4). KEGG classifies different metabolic pathways from three levels of level 1, level 2, and level 3. Figure 4 is showing the differences of metabolic pathways of different reactors at level 2 and level 3. The ordinates are different metabolic pathways, and the abscissa is the proportion of various metabolic pathways in the metatranscriptome. The top three transcriptional at level 2 pathways were ‘Energy metabolism’, ‘Global and overview maps’, and ‘Carbohydrate metabolism’. The relative abundance of ‘Energy metabolism’ in Control, Fe group, and Ni group were 15.9%, 10.2% and 14.9%, respectively. The results indicated the importance of ‘Energy metabolism’ function for acetate production of MES, as inferred from the essential intracellular electron transfer that accompanied by energy metabolism. The top 5 expression categories at level 3 pathways in ‘Global and overview maps’, ‘Energy metabolism’ and ‘Carbohydrate metabolism’ were shown in Fig. 4b. The dominant expressed categories at level 3 pathways in ‘Global and overview maps’ were ‘Carbon metabolism’, and ‘Biosynthesis of amino acids’, which are the basic metabolism for the survival and growth of microorganism. In ‘Energy metabolism’, ‘Carbon fixation pathways in prokaryotes’ was one of the dominant at level 3 pathways, which was consistent with the performance of the MES reactors for CO2 fixation and acetate production. ‘Carbon fixation pathways in prokaryotes’ includes the WL pathway, which is the primary pathway for CO2 fixation in MES, and the proportion of ‘Carbon fixation pathways in prokaryotes’ transcriptional expression was altered by the addition of Fe2+ (1.9%) and Ni2+ (2.3%), compared to that of Control (2.2% ). The high expression of “methane metabolism” was consistent with the predicted high expression genus of “Methanobacterium” (Fig. 3). The expression abundance of “Methane metabolism” in the control group was much higher than that of Fe group and Ni group.
The distribution of transcripts to general functional categories was assessed based on the BLAST against the EggNOG database. ‘Energy production and conversion’ was also one of the top abundant expressed COG functional annotation (Figure S3). The order of expression abundance of ‘Energy production and conversion’ in the three groups based on COG annotation was consistent with the order of ‘Energy metabolism’ based on KEGG annotation, which mutual verified the reliability of the annotations.
Go Enrichment Analysis Of Degs
Total of 49212 DEGs between the Fe or Ni group and the control were identified. The volcano maps of the identified DEGs are shown in Figure S4. Compared to the control, 6633 genes up-regulated and 4496 genes down-regulated with ferrous ion addition, and the proportion of significantly up-regulated and down regulated genes were 13.5% and 9.1% of the total genes. Meanwhile, 6365 genes up-regulated and 4879 genes down-regulated with nickel ions addition, and the proportion of significantly up-regulated and down regulated genes were 12.9% and 9.9% of the total genes.
The top 25 enriched GO categories with Fe2+ and Ni2+ addition were shown in Fig. 5. Go functional classification is mainly divided into three parts, Molecular Function (MF), Biological Process (BP) and Cellular Component (CC). The addition of Fe2+ and Ni2+ were a certain enrichment effect on GO function, and Fig. 5 is showing the enrichment of GO categories. The ordinates are different GO categories, the abscissa is the enrichment proportion of GO categories, and the bubble size in the figure is the expression quantity of GO categories in the metatranscriptome. The genes were mainly classified in ‘molecular function’ in both Fe group and Ni group, and the enrichment ratio of Ni group was slightly higher than that of Fe group. The common enriched gene categories in Fe and Ni group were peptidase activity, acting on L-amino acid peptides (GO:0070011), peptidase activity (GO:0008233), oxidoreductase activity (GO:0016491), hydrolase activity (GO:0016787), catalytic activity (GO:0003824), anion binding (GO:0043168), ion binding (GO:0043167), molecular function (GO:0003674), purine nucleotide binding (GO:0017076), small molecule binding (GO:0036094), and nucleotide binding (GO:0001882). Thereinto, the oxidoreductase activity and catalytic activity were the catalysis function of an oxidation-reduction in biochemical reactions, which were related to electron transfer. In the Ni group, the enrichment ratio of oxidoreductase activity and catalytic activity were 19.5% and 15.8%, respectively, higher than that of 15.9% and 14.2% in the Fe group. The addition of Ni2+ promoted the enrichment of oxidoreductase activity and catalytic activity in MES, which was conducive to electron transfer and energy conversion.
Expression Differences Of Genes In Energy Conservation System And Wood–ljungdahl (Wl) Pathway
Expression differences of main genes for energy conversion are shown in Fig. 6, with the definition of analyzed KOs shown in Table S2. In the KEGG database, energy conversion-related KOs were discovered and examined. Figure 6 is showing the impact of Fe2+ or Ni2+ addition on the expression of typical energy conversion complex KOs. The ordinates are the KOs of energy conversion complex, and the abscissa is the log2 FC in the metatranscriptome. H2ase is an important energy metabolism enzyme, which use electron bifurcation to drive the endergonic ferredoxin reduction by coupling it to the exergonic NAD+ reduction (Lohner et al. 2014; Feng et al. 2022; Tai et al. 2021; Schuchmann and Muller 2012). In the Fe group, a large part of H2ase KEGG Orthogroups (KOs) met the standard (\(FDR<0.05\) and \(\left|\text{l}\text{o}\text{g}2 \text{F}\text{C}\right|\ge 1\)), such as K03618 (hydrogenase-1 operon protein HyaF), K03620 (Ni/Fe-hydrogenase 1 B-type cytochrome subunit), K04651 (hydrogenase nickel incorporation protein HypA/HybF), K04654 (hydrogenase expression/formation protein HypD), and K05796 (electron transport protein HydN) (Fig. 6a). The log2 FC of K03618, K03620, K04651, K04654, and K05796 in Fe group were 4.6, 2.8, 5.5, 1.6, 1.1, and 2.3, respectively. Moreover, in the Ni group, more H2ase KOs met the standard compare with Fe group, such as K00437 (Ni-Fe hydrogenase large subunit), K03618, K04651, K04653 (hydrogenase expression/formation protein HypC), K05586 (bidirectional Ni-Fe hydrogenase diaphorase subunit, hoxE), K05587 (bidirectional Ni-Fe hydrogenase diaphorase subunit, hoxF), K05588 (bidirectional Ni-Fe hydrogenase diaphorase subunit, hoxU), K05922 (quinone-reactive Ni/Fe-hydrogenase large subunit), and K05927 (quinone-reactive Ni/Fe-hydrogenase small subunit), with log2 FC of 2.8, 2.2, 4.1, 1.1, 3.6, 1.4, 3.0, 5.1, and 4.9, respectively. ATPase is a membrane-bound electron transport system, with sodium pump as the motive force (Mayer and Muller 2014). The expression of K02110 (F-type H+-transporting ATPase subunit c) and K02114 (F-type H+-transporting ATPase subunit epsilon) in ATPase were promoted, and the expression of K01535 (H+-transporting ATPase), K02107 (V/A-type H+/Na+-transporting ATPase subunit G/H), K02108 (F-type H+-transporting ATPase subunit a), K02119 (V/A-type H+/Na+-transporting ATPase subunit C), K02120 (V/A-type H+/Na+-transporting ATPase subunit D), K02121 (V/A-type H+/Na+-transporting ATPase subunit E), K02123 (V/A-type H+/Na+-transporting ATPase subunit I), and K06865 (ATPase) were decreased with ferrous ion addition(Fig. 6b). While in Ni group, the expression of K02108, K02110, K02111 (F-type H+/Na+-transporting ATPase subunit alpha), K02112 (F-type H+/Na+-transporting ATPase subunit beta), K02113 (F-type H+-transporting ATPase subunit delta), K02114, and K02115 (F-type H+-transporting ATPase subunit gamma) were promoted, only K01535 and K02116 (ATP synthase protein I) expression were depressed. NADH oxidoreductase complexes are essential cofactors for metabolism and intercellular electrons transfer in microorganisms. The expression of K00340 (NADH-quinone oxidoreductase subunit K), K00356 (NADH dehydrogenase) and K13378 (NADH-quinone oxidoreductase subunit C/D) in NADH oxidoreductase complex were promoted with ferrous ion addition, while the expression of K00330 (NADH-quinone oxidoreductase subunit A), K00333 (NADH-quinone oxidoreductase subunit D) and K00343 (NADH-quinone oxidoreductase subunit N) were depressed. While, the nickel ions addition promoted the expression of K00338 (NADH-quinone oxidoreductase subunit I) and K13378. Rnf complex is important in energy conservation in acetogens (Zhu et al. 2020; Tremblay et al. 2013). The expression of K03613 (H+/Na+-translocating ferredoxin: NAD+ oxidoreductase subunit E) in Rnf complex was promoted in the Fe group. While, the nickel ions addition depressed the expression of K03612 (H+/Na+-translocating ferredoxin: NAD+ oxidoreductase subunit G) in the Ni group.
The KOs of theWL pathway was found in the metatranscriptomic libraries in all samples. The differentially expressed KOs with \(FDR<0.05\) and \(\left|\text{l}\text{o}\text{g}2 \text{F}\text{C}\right|\ge 1\) are shown in Fig. 7, with the definition of analyzed KOs shown in Table S3. Figure 7 is showing the impact of Fe2+ or Ni2+ addition on the expression of WL pathway KOs. The ordinates are the KOs of WL pathway, and the abscissa is the log2 FC in the metatranscriptome. In Fe group, the log2 FC of K05299 (formate dehydrogenase (NADP+) alpha subunit) and K15023 (5-methyltetrahydrofolate corrinoid/iron sulfur protein methyltransferase) was 2.6 and 3.1. While, in Ni group, the log2 FC of K00198 (anaerobic carbon-monoxide dehydrogenase catalytic subunit), K05299, K15022 (formate dehydrogenase (NADP+) beta subunit), and K15023 was 1.5, -2.0, 2.1 and 1.4, respectively.